the nonclassical hla - g molecule presents several properties that differ from other classical class i hla ( -a , -b , and -c ) molecules , including restricted tissue distribution ; limited protein variability ; presence of several membrane - bound and soluble isoforms ; unique molecular structure , presenting a particular peptide - binding groove that impairs peptide presentation to t cells ; ability to form dimers and polymers and a reduced cytoplasmic tail that impairs molecule turnover ; and , most importantly , the molecule that modulates several functions of immune system cells ( reviewed by ) .
the interaction of hla - g with leukocyte receptors , particularly ilt-2 and ilt-4 , downregulates the cytotoxic activity of t cd8 and natural killer cells and inhibits antigen presentation and lymphocyte proliferation [ 1 , 2 ] .
due to all of these properties , hla - g has been recognized as a tolerogenic molecule , and the tissue expression of hla - g may protect or harm ; that is , it may protect allografts against attack by the recipient immune system and may impair the cytotoxic immune response against tumor cells .
the coding region exhibits few polymorphic sites randomly distributed along exons and introns , contrasting with the high rate of exonic polymorphic sites observed in classical hla class i genes .
the exonic nucleotide sequences encoding residues that are important for molecule dimerization and molecule interaction with leucocyte receptors are usually conserved , indicating that the overall structure of the molecule was maintained throughout human evolution [ 1 , 2 , 4 , 5 ] . considering that hla - g is expressed on the surface of placenta trophoblast cells , allowing the fetus to properly develop despite the maternal immune response , some sort of functional conservation was expected . on the other hand , gene regulatory regions present several polymorphic sites close to nucleotide sequences that serve as gene regulatory elements [ 69 ] .
nucleotide variability in the promoter region may influence hla - g levels by modifying binding affinity for transcription factors .
in contrast to classical hla class i genes , the promoter region of hla - g does not have responsive elements for ifn- or nf-b .
similarly , nucleotide variability at the 3 untranslated region ( 3utr ) may influence hla - g mrna stability , microrna targeting , or both , affecting the posttranscriptional gene regulation . considering that the structure of hla - g molecules has been maintained throughout evolution , the quantity of produced molecules may primarily depend on factors that modulate gene expression by transcriptional and posttranscriptional mechanisms .
firstly , we will review the structure of the hla - g promoter region and its implication in transcriptional gene control ; secondly , the structure of the hla - g 3utr and the major actors of the posttranscriptional gene control ; and , finally , the presence of elements in the coding region that may regulate gene expression and differential mrna splicing .
there is no consensus regarding the positions of the nucleotide variation in the hla - g promoter and 3utr , mainly because ( i ) the imgt / hla database only presents sequences within 300 bases upstream to the first translated atg , ( ii ) the complete 3utr gene segment is not considered in the imgt database , and ( iii ) several hla alleles were described presenting only some exon sequences .
therefore , the nucleotide positions used in the present study follow the one presented in the ng_029039 sequence ( http://www.ncbi.nlm.nih.gov/nuccore/ng_029039 ) . the nucleotide named as + 1 is the adenine of the first translated atg ( position 5867 at ng_029039 ) .
variations within regulatory elements in the upstream 5 untranslated region and 5 promoter were denoted as negative values , considering position 5866 at ng_029039 as nucleotide 1 .
the hla class i genes are usually very similar in nucleotide sequence and structure because most of these genes have been generated in a series of imperfect duplications . therefore , in general , the same regulatory elements are acting in hla class i genes , with some differences for each hla class i locus . the hla class i promoters
are usually conserved , presenting cis - acting regulatory elements mainly within 220 bases upstream to the first translated atg
. however , the hla - g promoter is atypical compared to other hla class i genes since most of these regulatory elements are not functional .
the hla - g locus presents a tissue - restricted expression pattern , being expressed in physiological conditions only in certain tissues such as trophoblast at the maternal - fetal interface , thymus , cornea , pancreas , proximal nail matrix , erythroblast , and mesenchymal stem cells [ 1 , 1118 ] . in view of the immunomodulatory properties of the hla - g molecule
overall , hla class i genes present two main regulatory modules in the proximal promoter region ( 200 bases upstream to the translation start point ) , including ( i ) the enhancer a ( enha ) combined with an interferon - stimulated response element ( isre ) and ( ii ) the sxy module , in which the transcription apparatus is mounted ( figure 1 ) [ 1924 ] .
however , these regulatory elements present locus - specific differences leading to different levels of hla class i constitutive- and induced - expression ( reviewed at [ 24 , 25 ] )
. the enha element includes two adjacent palindromic nf-b binding sites ( b1 and b2 ) that interact with the nf-b family of transcription factors , both important to the constitutive and/or induced expression of hla class i genes .
this family includes several members , such as p50 , p65 ( also known as rela ) , p52 , c - rel , and relb , all usually acting by forming homo- or heterodimers . theoretically , the interaction of these factors with the enha element may transactivate ( acting on any b binding site ) any hla class i gene .
thus , the hla locus - specific transcription rate would be determined by ( i ) the levels of nf-b / rel family proteins in different tissues , ( ii ) modifications in the regulatory sequences , and ( iii ) potential activation of different nf-b / rel dimers . in addition , enha may be a target sequence for other dna - binding proteins , such as proteins of the leucine zipper transcription factor family .
for instance , p65 has a potent transactivation domain and might operate as a p65/p50 heterodimer or p65/p65 homodimer , while p50 lacks this transactivation domain and may not transactivate as a p50/p50 homodimer .
enha also mediates the tnf - induced transcription of hla class i molecules [ 20 , 29 ] .
due to variations in the enha nucleotide sequences among different hla class i genes , nf-b / rel factors may interact as homo- or heterodimers resulting in different transcription levels .
the hla - g enha element ( including b1 and b2 sites ) encompasses nucleotides 198 and 172 ( regarding ng_029039 ) and , compared to other hla class i genes , it is the most divergent one [ 19 , 20 ] .
in fact , the b - sites in the hla - g promoter ( enha ) are reported to bind only p50/p50 homodimers   ( figure 1 ) . as presented earlier ,
thus , although hla - g possesses an nf-b responsive element , it is not as efficient as the hla class i classical genes .
isre is a target site for the interferon regulatory factor family , including the interferon regulatory factor-1 ( irf-1 , activator ) , irf-2 , and irf-8 ( inhibitors ) .
interferon- ( ifn- ) is the most potent cytokine inducing hla class i gene expression .
ifn- induces the expression of irf-1 by the activation of the janus kinases ( jak ) 1 and 2 and phosphorylation of stat1 ( jak / stat pathway ) [ 19 , 21 ] .
isre is adjacent to the enha element ( constituting the module enha / isre presented earlier ) and , thus , isre and enha regulate hla class i expression cooperatively ( figure 1 ) .
isre also participates in the transactivation of 2-microglobulin , which is associated with the heavy  chain of the hla class i molecule , and this information is important because an unbalanced production of these chains may impair correct hla molecule assembly .
the nucleotide sequence of isre also varies among hla class i loci . in this respect ,
locus - specific differences were observed in the ifn - induced expression levels of hla class i genes [ 21 , 2931 ] .
the hla - a locus , for instance , does not respond to ifn at the same level as hla - b and hla - c , probably because of differences in the isre structure [ 19 , 21 , 2931 ] .
however , when comparing isre of the hla - g locus with other class i genes , encompassing nucleotides 171 to 161 , the hla - g gene presents the most divergent isre compared to the isre consensus sequence , followed by hla - e [ 19 , 21 ] , raising the issue of whether or not this element is fully functional for hla - g and hla - e .
in fact , neither hla - g nor hla - e isres mediate ifn--induced transactivation , and the binding of irf-1 is not detected for hla - g .
however , in the same way , probably because of the defective nature of the hla - g isre , the binding of irf-2 ( transcription repression ) was also not detected for hla - g .
isre is also a target for other protein complexes that may mediate hla class i transactivation .
however , hla - gb2 ( enha ) and isre seem to bind only the constitutively expressed factor sp1 ( also known as specificity protein 1 ) [ 21 , 25 ]
. nevertheless , the binding of sp1 does not modulate the constitutive or ifn - induced transactivation of hla class i genes , including hla - g . on the other hand , a candidate interferon - gamma activated site ( gas )
was described between the 741 and 733 positions , presenting a sequence that would be compatible with a gas consensus sequence ( figure 1 ) .
however , besides this new candidate , ifn- , ifn- , and ifn- treatments failed to increase hla - g expression , a fact that was accredited to the defective nature of both the new candidate and the enha / isre region [ 3234 ] .
nonetheless , another study showed that ifn- enhances hla - g expression by another isre present next to the nonfunctional gas element at positions 754 to 743 .
the sxy module comprises the s , x1 , and x2 ( also known as site  ) boxes and the y box ( also known as the enhancer b or ccaat box ) .
the x1 box is a target for the multiprotein complex regulatory factor x ( rfx ) , including the rfx5 , the regulatory factor x - associated protein ( rfxap ) , and rfxank [ 20 , 25 , 35 , 36 ] .
these rfx members have been shown to interact with the class ii transactivator ( ciita ) [ 37 , 38 ] , which is also an important element for hla class i gene transactivation .
the x2 box is a binding target for the atf / creb ( activating transcription factor / camp response element binding protein ) transcription factor family .
box y is a binding target for nuclear factor y ( nfy ) , including subunits alpha ( nfya ) , beta ( nfyb ) , and gamma ( nfyc ) [ 25 , 40 ] .
the binding of these factors to the sxy module allows the further binding of the coactivator ciita and the nod - like receptor family card domain containing 5 ( nlrc5 ) factors [ 25 , 41 , 42 ] .
the ciita is constitutively expressed by antigen presenting cells and is induced by ifn- , and it transactivates hla class i genes [ 41 , 43 ] .
nlrc5 transactivates hla class i genes ( but not hla class ii ) and is constitutively expressed in a series of different tissues , mainly hematopoietic cells , or is induced by inf- [ 4446 ] . for hla - g , the sxy module presents sequences compatible only with the s and x1 elements , but divergent from x2 and y elements ( figure 1 ) .
therefore , ciita , which is dependent on a functional sxy module , does not transactivate hla - g , mainly because of the missing x2 and y elements [ 25 , 41 , 42 , 47 , 48 ] .
considering all the elements discussed above , it became clear that the hla - g proximal promoter ( within 200 bases ) did not mediate transactivation by the principal hla class i transactivation mechanisms .
in addition , studies evaluating the hla - g promoter region within 1438  bp from atg did not detect differences in the basal level of transactivation for different hla - g promoters in different cell types [ 49 , 50 ] .
some alternative regulatory elements within the hla - g gene promoter have been described . a heat shock element ( hse ) which would respond to the presence of heat shock proteins ( hsp ) , especially the heat shock factor 1 ( hsf1 ) ,
hla - g transcription is induced by heat shock ( physical stress ) or arsenate treatment ( chemical stress ) in human melanoma and glioblastoma cell lines , in which stress - induced hsf1 binds to an hse lying between the 464 and 453 positions .
this hse response was detected for hla - g but not for other hla class i genes .
hla - g expression may also be induced by progesterone , which is an immunomodulatory steroid hormone secreted both by the corpus luteum and placenta , allowing endometrium maintenance and embryo implantation .
the mechanism underlying this induction is primarily mediated by the activation of the progesterone receptor ( pr ) and its subsequent binding to an alternative progesterone response element ( pre ) found in the hla - g promoter between positions 52 and 38 , overlapping the hla - g tata box   ( figure 1 ) .
experiments with transgenic mice allowed the identification of a locus control region ( lcr ) candidate located at least 1.2  kb upstream to the first translated atg .
this region is critical for the hla - g expression regarding when and where it should be expressed .
it is possible that this region acts by maintaining the chromatin in an open state or active configuration , enhancing gene expression [ 54 , 55 ] .
in addition , it may bind protein complexes associated with activation and inhibition of hla - g transcription [ 56 , 57 ] .
at least three cre / tre candidate sites ( camp response element / tpa response element ) have been already considered , the first one being situated between the 1387 and 1371 positions ( inside the putative lcr region discussed earlier ) , the second between the 941 and 935 positions , and the third between the 777 and 771 positions ( figure 1 ) . the first cre site ( at lcr )
was described to be an in vitro target site for c - jun by using electrophoretic mobility shift assay ( emsa ) .
c - jun , together with c - fos , forms the ap-1 early response transcription factor .
in addition , this same site was reported to bind atf1/creb1 in vitro and in situ by using chromatin immunoprecipitation ( chip ) .
the second cre / tre site binds in vitro to creb1 and the third site binds in vitro to atf1/creb1 .
mutations in all three cre / tre sites have been reported to reduce the hla - g creb1 transactivation , but a stronger inhibition was observed when the first cre / tre site ( at the lcr ) was mutated .
the repressor factor rreb1 ( ras responsive element binding 1 ) may also be implicated in hla - g expression regulation .
at least three binding sites for rreb1 , known as ras response elements ( rre ) , in the hla - g promoter region have been described .
the consensus sequence ggtcct , corresponding to one of the binding sites for rreb1 , was found in the proximal promoter between the 59 and 54 positions ( one direct site ) and between the 148 and 143 positions and the 139 and 134 positions ( a direct site and an inverted site ) . a target site related to the other consensus - binding site for rreb1 , ccccaccatcccc , was found within the lcr between the 1363 and 1358 positions ( figure 1 ) .
the mechanism underlying rreb1 repression is probably associated with the recruitment of the corepressor c - terminal binding protein 1 or 2 ( ctbp-1 or ctbp-2 ) , or both , and the deacetylase 1 ( hdac1 ) , which is involved in chromatin remodeling probably increasing chromatin condensation and hampering transcription factor accessibility [ 58 , 59 ] .
the gli-3 repressor , a signal transducer of the hedgehog pathway ( hh ) , has also been reported to regulate hla - g during the maturation of osteoblasts , especially in the production of the hla - g5 isoform .
it acts by a direct interaction of the hh signaling transducer factor gli-3 with the hla - g promoter .
however , it is not clear whether the hh signaling pathway , a highly conserved molecular pathway involved in the development of several tissues , directly regulates hla - g5 expression in other cell types .
a negative regulator of gene expression is observed in a sequence about 4  kb upstream to the hla - g translation starting point , overlapping with a line-1 sequence   ( figure 1 ) .
lines ( long interspersed elements ) are a group of retrotransposons , which are highly repetitive elements from the eukaryotic genome that contribute to genome variability .
the line-1 element described for hla - g ( named gl ) is an at - rich sequence ( about 60% ) that presents more sites with a high probability of forming hairpin loops than the general line sequence .
these hairpin loops might directly or indirectly interact with the hla - g promoter and interfere with the binding of transcriptional factors and enhancers .
line elements are frequently found lying in the 5 upstream regulatory region of other hla class i genes , including hla - a .
however , the line sequence found in the hla - a promoter ( named al ) is not transcriptionally active and is shorter than the one found in hla - g ( gl ) .
therefore , the presence of this gl element in the hla - g promoter would explain its limited expression compared with other class i genes .
however , it should be noticed that this gl element is also present in hla - g - expressing cells ; thus , other regulatory elements might inhibit or overcome this negative regulation .
hypoxia is an important physiological microenvironment for placentation and for the formation of the maternal - fetal interface .
the microenvironment is also crucial for the function of t and b cells . in this scenario ,
the hypoxia - inducible factor ( hif ) is involved in the control of cellular responses to oxygen depletion . the hla - g expression ( membrane and soluble )
is 2 times increased when extravillous cytotrophoblasts are cultivated under only 2% oxygen . likewise , hypoxia is associated with increased hla - g transcription in a series of hla - g - negative tumor lineages , such as 1074mel [ 64 , 65 ] and m8 . a consensus hypoxia response element ( hre )  
some inducers of hla - g expression have been described ; but the underlying induction mechanisms are unknown .
interleukin 10 ( il-10 ) , which is produced by lymphocytes , monocytes , macrophages , placenta , and some tumors , may induce hla - g expression and the downregulation of other hla class i and ii genes [ 6870 ] .
cortisol , a glucocorticoid produced by the adrenal gland , is a potent immunomodulatory hormone at high doses .
hla - g expression in trophoblastic cells was increased following treatment with dexamethasone or hydrocortisone , but no complete glucocorticoid response element ( gre ) has been identified in the hla - g promoter .
granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a protein secreted by macrophages , t cells , mast cells , nk cells , endothelial cells , fibroblasts , and uterine epithelium .
gm - csf increases hla - g expression when combined with inf- treatment , but no effect is observed for gm - csf alone [ 72 , 73 ] . the leukemia inhibitory factor ( lif ) is a cytokine expressed at the maternal - fetal interface in the cytotrophoblast that plays an important role in implantation .
lif is mainly expressed in the implantation window . by using the choriocarcinoma cell line jeg3 ,
it was demonstrated that lif induces full - length membrane hla - g ( hla - g1 ) expression on the jeg3 cell surface .
in addition , lif may induce hla - g1 expression in the presence of erap1 ( endoplasmic reticulum aminopeptidase-1 ) expressed in the endoplasmic reticulum .
repression of erap1 in jeg3 cells treated with lif diminishes hla - g expression , suggesting a role for erap in hla - g regulation .
some drugs may also induce hla - g production , such as methotrexate ( mtx ) , one of the most used antirheumatic drugs for the treatment of rheumatoid arthritis ( ra ) .
mtx can induce apoptosis of mitogen - stimulated peripheral blood mononuclear cells ( pbmcs ) resembling the mechanisms underlying the inhibition of cytotoxic t cd8 + cell activity by soluble hla - g molecules .
mtx can induce the production of shla - g in unstimulated ra or healthy individual pbmcs and may have a role in the clinical outcome of ra patients .
the mechanisms underlying shla - g production after mtx treatment are unknown , but it was reported that mtx therapy mediates an increase of interleukin-10-producing cells , which in turn may stimulate hla - g production .
data from the 1000 genomes project , including 1092 individuals from 14 different populations , showed 32 variable sites within 1500 nucleotides upstream to the first translated atg .
most of these variable sites have been already described in other populations or samples different from those evaluated by the 1000 genomes consortium [ 69 , 7781 ] . of those ,
24 variable sites present frequencies higher than 1% and 14 present frequencies higher than 10% in the global 1000 genomes data ( all 1092 individuals ) .
these variable sites may be important for the regulation of hla - g expression and may act in different ways .
polymorphisms in the proximal promoter of paan - ag , the functional homologue of hla - g in the olive baboon , have been shown to influence nf-b binding and transcription activity [ 82 , 83 ] . however , the human variable sites may act by mechanisms differing from those described above because , generally , these variable sites do not coincide with known regulatory elements ( figure 1 ) .
variation in regulatory elements may affect the binding of the corresponding regulatory factors . in this respect ,
only four variable sites coincide with known regulatory elements : ( i ) position 1377 in the first cre site of the lcr , ( ii ) positions 1310 and 1305 of the lcr , and ( iii ) position 56 of the ras response element ( rre ) in the proximal promoter . of these
, only the ones at positions 1305 and 56 are frequently found worldwide ( figure 1 ) .
other variable sites are close to known regulatory elements and may somehow influence the binding of transcription factors . in this group
we may observe variable sites at positions 762 ( between a cre and isre ) , 725 ( next to a nonfunctional gas element ) , 477 and 433 ( around the hse ) , and 201 ( next to enhancer a ) ( figure 1 ) .
the variable site at position 725 , in which the minor allele ( g ) is present in 9.8% of the chromosomes evaluated in the 1000 genomes project , was associated with differential hla - g expression .
hla - g promoter haplotypes ( between 1389 and 55 and not considering primer sequences ) were cloned into luciferase expression vectors and transfected to the hla - g expressing cell jeg-3 , resulting in a significantly higher expression level of the promoters presenting guanine at position 725 .
in addition , another study described the same influence of position 725 on hla - g expression levels .
this same polymorphism ( 725 g ) has been reported to be associated with sporadic miscarriage   and end - stage renal disease , while the most frequent allele ( 725 c ) has been reported to protect against multiple sclerosis . nevertheless , despite the lack of studies regarding hla - g promoter polymorphisms and hla - g expression ,
the polymorphism at position 964 , which is very frequent among the populations evaluated by the 1000 genomes consortium , was associated with asthma .
the 964 g / g genotype was associated with asthmatic children of affected mothers , whereas the a / a genotype was associated with asthmatic children of unaffected mothers .
the 964 a and 486 c alleles , together with the 725 g allele , were also associated with protection against end - stage renal disease .
the polymorphism at position 1305 , also very frequent among the 1000 genomes populations , was associated with nonsegmental vitiligo .
the methylation status of the hla - g promoter is also very important to the hla - g transcriptional activity .
it has been reported that the cpg islands in the hla - g promoter region of jar ( choriocarcinoma ) cells , which does not express hla - g , were fully methylated , whereas for an hla - g expressing cell such as jeg-3 , the cpg islands were only partially methylated [ 91 , 92 ] .
in addition , hla - g expression was induced in several tumor cell lines by using demethylation agents , such as 5-aza-2deoxycytidine [ 9397 ] . moreover ,
the levels of histone acetylation in the hla - g promoter chromatin have been reported to be significantly enhanced in fon+ ( melanoma ) and jeg-3 ( human placental choriocarcinoma cell line ) cell lines , both expressing hla - g , while in non - hla - g expressing cell lines , such as m8 ( melanoma ) and jar , histones seem to be hypomethylated [ 94 , 96 , 98 ] .
histone acetylation is usually associated with a relaxed chromatin structure , therefore , with greater levels of gene expression [ 99 , 100 ] . in this respect ,
polymorphisms in the hla - g promoter , especially in the cpg islands , might also be associated with different methylation profiles .
although most of the hla - g promoter variable sites do not occur inside known regulatory elements ( figure 1 ) , balancing selection has been reported to maintain divergent haplotypes in the 5 promoter [ 6 , 8 , 9 , 78 , 79 ] and 3utr regulatory regions [ 6 , 27 , 28 , 78 , 101 , 102 ] .
in fact , at least 14 variable sites in the promoter region do present frequencies higher than 10% , and 11 variable sites present frequencies higher than 44% ( figure 1 ) . however , considering the haplotypes described for the hla - g promoter , which seem to be the same worldwide [ 6 , 8 , 9 , 7779 ] , most of these frequent variable sites are in complete linkage disequilibrium ( ld ) , and just four main hla - g promoter lineages are associated with these variable sites .
these promoter lineages were first proposed by ober 's group   and subsequently confirmed and named in a brazilian study as promo - g010101 , promo - g010102 , promo - g0103 , and promo - g0104 .
in addition , considering data from the 1000 genomes project , only nine promoter haplotypes present frequencies higher than 1% in worldwide populations ( figure 2 ) , but two of them , promo - g010101a and promo - g010102a , which are the most divergent ones , account for more than 60% of all haplotypes . nevertheless , despite the fact that most of these frequent hla - g variable sites are not within known regulatory elements , several lines of evidence indicate balancing selection acting on the hla - g promoter found in several populations [ 6 , 8 , 9 , 78 , 79 ] , suggesting that divergent promoters have been maintained with high heterozygosis .
this observation is probably related to a possible better fitness of individuals carrying both high- and low - expressing promoters .
therefore , these divergent hla - g promoter haplotypes are probably associated with differential hla - g expression , but the mechanisms are unknown . however , as discussed later , the pattern of ld observed for the promoter region extends up to hla - g 3utr [ 6 , 8 , 9 , 27 , 7779 ] and at least 20  kb beyond the hla - g 3utr .
thus , selective pressures acting on other hla - g regions as well as adjacent sequences might also influence hla - g promoter variability and heterozygosis .
as previously stated , there is no consensus regarding the positions of the nucleotide variation in the hla - g 3utr , considered to be located mostly in exon 8 .
since there is no official information regarding the hla - g 3utr sequences , the nucleotide positions used in the present study follow those previously reported by our group [ 1 , 6 , 27 ] , that is , inferring polymorphic sites in 3utr using the original hla - g sequence described by geraghty and colleagues   and considering nucleotide + 1 as the adenine of the first translated atg ( similar to the imgt / hla description ) . in the hla - g 3utr , there is a well - studied polymorphism that consists of a 14-nucleotide deletion ( rs371194629 or rs66554220 ) , also known as the 14-bp indel ( insertion / deletion ) polymorphism .
the sequence used as a model for the hla - g promoter structure ( ng_029039 ) does not present this 14-nucleotide sequence ( that would be inserted between nucleotides + 2960 and + 2961 ) . given that the presence of these 14 nucleotides is also found in gorillas and chimpanzees , it should represent the ancestor allele , and the 14 bp sequence
therefore , any position after nucleotide + 2960 is taken considering the original ng_029039 sequence plus 14 bases .
for instance , the polymorphism at the + 3142 position discussed later in this review refers to the + 3128 nucleotide in the ng_029039 reference sequence . due to a premature stop codon ( positions + 2536 to + 2538 in ng_029039 ) , the hla - g gene presents a relatively large 3utr genomic sequence that extends up to the + 3292 nucleotide , encompassing approximately 754 nucleotides . inside the 3utr genomic region
, there is an intron that is spliced out , giving rise to the mature hla - g mrna with a 3utr sequence of approximately 397 nucleotides ( considering the presence of the 14 bases discussed earlier ) .
this 3utr is a key feature for transcriptional hla - g regulation , which is important for ( i ) hla - g mrna stability , ( ii ) targeting specific micrornas , and ( iii ) polyadenylation signal in the au - rich regulatory mrna element .
the mrna availability for translation , as well as consequent protein production and maturation , is constantly balanced by the opposing forces of transcription levels and mrna decay .
the transcription level is mainly driven by the 5 ' regulatory region and the presence of specific transcription factors , while mrna decay is mainly driven by its intrinsic stability ( which is dependent on the nucleotide sequence ) and the action of micrornas .
micrornas may negatively regulate gene expression by translation suppression , rna degradation , or both [ 104 , 106108 ] .
the first mirna was reported in 1993 , and more than 2000 human micrornas have been reported to date [ 110 , 111 ] .
the hla - g 3utr presents several polymorphic sites , some of which have been associated with differential hla - g expression profiles .
although the hla - g 3utr segment is quite short compared to the same region in other genes , it presents at least eight polymorphic sites that are frequently found in worldwide populations ( figure 3 ) .
the hla - g 3utr variability and haplotypes were systematically explored in a southeastern brazilian population , in which seven frequent haplotypes were described , encompassing these eight polymorphic sites , designated utr-1 to utr-7 , and a rare one named utr-8 .
the relationship between hla - g 3utr polymorphisms ( especially for the 14 bp polymorphism ) and other variable sites in the hla - g coding and promoter region was also previously explored [ 77 , 78 , 105 , 112 , 113 ] .
furthermore , several populations were evaluated regarding these polymorphic sites , including additional samples from other brazilian regions and other worldwide populations , and the same pattern of 3utr variability has been observed [ 6 , 27 , 28 , 85 , 102 , 114121 ] .
recently , the variability at the hla - g locus was explored by using the 1000 genomes data [ 28 , 102 ] and , taking together all of these studies in the last decade , it became clear that the same 3utr pattern observed in brazilians   is found worldwide , with just some new low frequency haplotypes .
most of the polymorphisms present in the hla - g 3utr may influence the hla - g expression profile by different mechanisms .
since they are present in a short mrna sequence with just some nucleotides apart , and since the pattern of haplotypes is quite conserved [ 28 , 102 ] , the influence of each polymorphic site on the hla - g expression profile may not be independent of other polymorphic sites ; that is , extended haplotypes should be considered due to the cumulative effects of different polymorphisms .
for example , the + 3003 , + 3010 , + 3027 , and + 3035 polymorphic sites encompass only 32 nucleotides that are also in linkage disequilibrium with each other and in linkage disequilibrium with variable sites in the coding and promoter segments   ( figure 3 ) .
the first hla - g 3utr polymorphic site associated with hla - g expression levels was an indel ( insertion / deletion ) variant known as the 14 bp polymorphism .
this polymorphism is characterized by the removal of a 14-nucleotide segment   between positions + 2961 and + 2974 , and it presents high frequency in all populations studied so far .
the ancestor allele ( the 14 bp presence or insertion ) is also found in gorillas and chimpanzees .
the 14 bp polymorphism has been associated with the magnitude of hla - g production [ 77 , 123125 ] , modulating hla - g mrna stability [ 113 , 126128 ] and also as a target for micrornas . in general , the presence of the 14-nucleotide sequence ( 5-atttgttcatgcct-3 ) has been associated with lower hla - g production for most membrane - bound and soluble isoforms in trophoblast samples [ 77 , 78 , 123 , 125 , 128 ] . however , svendsen and colleagues observed the opposite when k562 cells were transduced with ins-14  bp hla - g1 or with del-14  bp hla - g1 , in which the expression of hla - g1 was found to be higher for ins-14  bp cells compared to del-14  bp cells .
moreover , this 14-base sequence was also associated with an alternative splicing of the hla - g transcript , in which 92 bases from the mature 3utr hla - g mrna were removed ( including the 14-base sequence itself ) [ 113 , 128 ] , and these smaller transcripts were reported to be more stable than the complete transcript .
though influencing mrna stability , only a fraction of the mrna bearing these 14 nucleotides is further processed with the removal of 92 bases , and the greater stability apparently does not compensate for the lower hla - g levels associated with the 14-base sequence .
nevertheless , there are controversial results regarding the influence of this polymorphism in hla - g expression and alternative splicing .
the following four polymorphic sites , frequently found in the hla - g 3utr in worldwide populations , are present at positions + 3003 , + 3010 , + 3027 , and + 3035 [ 6 , 27 ] .
although no specific regulation mechanism has been described regarding these polymorphic sites , they might influence microrna binding .
additional polymorphic sites around this small hla - g 3utr segment are infrequently observed in worldwide populations , including the + 3001 c / t polymorphism observed in senegalese and northeastern brazilian populations [ 115 , 116 ] and the + 3033 c / g polymorphism observed among northeastern brazilians . although there are no studies evaluating the functional properties of these polymorphic sites , an in silico study reported that several micrornas might target this small segment . the nucleotide variation at position + 3142 has been associated with the magnitude of hla - g expression by posttranscription mechanisms , such as the interaction with micrornas .
it was functionally and computationally demonstrated that this variation site would influence the binding of specific micrornas , including mir-148a , mir-148b , and mir-152 .
the presence of a guanine at the + 3142 position increases the affinity of this region for these micrornas , hence decreasing hla - g expression by mrna degradation and translation suppression [ 106 , 129 , 130 ] .
this polymorphism , together with the 14-bp polymorphism , has been considered to be the most important one regarding hla - g posttranscription regulation , and methodologies have been proposed to quickly type these polymorphic sites [ 131 , 132 ] .
at least two studies have demonstrated that the + 3142 c / g polymorphic site may influence hla - g expression by modulating the mrna interaction with mir-152 , particularly in bronchial asthma [ 129 , 133 ] . however , there is no consensus regarding the influence of this polymorphic site on the binding of such micrornas , since another functional study did not detect this influence .
instead , it was reported that both mir-148a and mir-152 downregulate hla - g expression , irrespective of the + 3142 c or g alleles .
these micrornas have already been reported to modulate the expression of another classical hla class i gene , hla - c .
interestingly , only hla - c and hla - g are usually found at the maternal - fetal interface , indicating the presence of some sort of coordinated regulation .
similarly to mir-148a , mir-148b , and mir-152 , other micrornas have the potential to bind to the hla - g mrna 3utr and to influence hla - g expression .
the binding ability of these micrornas may be potentially influenced by polymorphisms observed in the hla - g 3utr .
another polymorphic site that has been associated with the magnitude of hla - g expression is located at position + 3187 a / g .
the mechanism underlying such association has been attributed to the proximity of this polymorphic site to an au - rich motif that mediates mrna degradation .
then , the presence of an adenine instead of a guanine at position + 3187 would lead to a decreased hla - g expression due to the increased number of adenines in this au - rich motif .
in addition to the micrornas that might target polymorphic sequences in the hla - g 3utr , some micrornas would bind to nonpolymorphic sequences and modulate hla - g expression irrespectively of the individual genetic background .
however , such approach has not yet been used and only micrornas targeting polymorphic sequences have been evaluated .
nevertheless , the microrna mir-133a was found to target a nonpolymorphic sequence upstream to the 14-b sequence fragment , between nucleotides + 2945 and + 2952 , downregulating hla - g expression ( figure 3 ) .
the conserved patterns of hla - g 3utr haplotypes and the few frequent haplotypes found worldwide [ 102 , 116 ] show that only one haplotype does carry all alleles that have been associated with high hla - g expression .
this haplotype , known as hla - g utr-1   ( 14 bp deletion/+3003 t/+3010 g/+3027 c/+3035 c/+3142 c/+3187 g/+3186 c ) , does not present the 14 bp sequence ; that is , it presents a 14 bp deletion , which was associated with highly soluble hla - g expression ; it presents a cytosine at position + 3142 ( less sensitive to specific micrornas targeting this region ) , and it exhibits a guanine at position + 3187 ( increased mrna stability ) . besides possessing these three polymorphic alleles associated with high hla - g production , utr-1 presents some other interesting features : ( i ) it is one of the most frequent 3utr haplotypes found worldwide , ( ii ) it has been described as one of the most recent hla - g 3utr haplotypes among the frequent ones due to its exclusive association with the presence of an alu element that is close to hla - g ( 20  kb downstream the 3utr ) , and ( iii ) utr-1 was recently associated with higher hla - g expression .
several studies have reported that the hla - g 3utr segment is also under selective pressures , whereby balancing selection is maintaining high levels of heterozygosis in this region [ 6 , 27 , 28 , 101 , 139 , 140 ] .
as observed worldwide [ 27 , 28 , 102 ] , the two most frequent hla - g 3utr haplotypes ( utr-1 and utr-2 ) are also the most divergent ones ( figure 3 ) .
therefore , the same phenomenon observed for the promoter region is also seen in the 3utr , in which high heterozygosis is observed between high- and low - expression haplotypes .
moreover , the rate of recombination in the hla - g locus is quite low , and the pattern of linkage disequilibrium found in the hla - g locus encompasses the promoter region , the coding region , the 3utr , and at least 20  kb downstream of the 3utr .
thus , in general , only few frequent extended haplotypes do exist and a specific promoter haplotype is usually accompanied by the same hla - g coding sequence and the same 3utr haplotype [ 68 , 28 , 77 , 78 , 102 ] .
the utr-1 haplotype , for example , is usually associated with the coding sequence for the hla - g*01:01:01:01 allele and the promo - g010101a promoter haplotype [ 68 , 28 , 102 ] .
therefore , the influence of each variable site at the hla - g transcriptional level must be considered .
the hla - g genetic structure resembles the class i structure , in which the first translated exon encodes the peptide signal , the second , third , and fourth ones encode the extracellular 1 , 2 , and 3 domains , respectively , and the fifth and sixth ones encode the transmembrane and the cytoplasmic domain of the heavy chain . considering the hla - g coding region ( from the first translated atg to the stop codon ) , at least 75 single nucleotide polymorphisms ( snp ) have been observed , defining the 50 currently described hla - g alleles , encoding only 16 distinct proteins ( imgt , database 3.14.0 , november 2013 ) .
similarly to what has been described for other genes such as irf4 , myc , ifng , and others [ 141146 ] , it is possible that intronic or exonic nucleotide sequences may exhibit affinity for transcription factors , thereby regulating the expression of the gene ; however , this subject has not yet been studied in the context of the hla - g gene .
the presence of certain polymorphic sites in the coding region may also regulate the expression of the seven described hla - g isoforms generated by alternative splicing of the primary transcript .
four of the hla - g isoforms are membrane - bound ( hla - g1 , g2 , g3 , and g4 ) and 3 are soluble ( g5 , g6 , and g7 ) ones .
hla - g1 is the complete isoform exhibiting a structure similar to that of the membrane - bound classical hla molecule , associated with 2-microglobulin , hla - g2 has no 2 domain , hla - g3 presents no 2 and 3 domains , and hla - g4 has no 3 domain .
the soluble hla - g5 and hla - g6 isoforms present the same extracellular domains of hla - g1 and hla - g2 , respectively , and the hla - g7 isoform has only the 1 domain [ 147149 ] .
in contrast to most of the currently described hla - g alleles that may produce all membrane - bound and soluble isoforms , the presence of stop codons in the coding region may yield truncated or missing hla - g isoforms .
the hla - g*01:05n null allele presents a cytosine deletion in the last nucleotide of codon 129 or in the first nucleotide of codon 130 ( exon 3 ) , leading to a tga stop signal in codon 189 , yielding incomplete formation of the hla - g1 , -g4 , and -g5 isoforms and normal expression of hla - g2 , -g3 , and -g7 [ 1 , 150 , 151 ] .
similarly , the hla - g*01:13n allele presents a c  t transition in the first base of codon 54 ( 1 domain ) , yielding the formation of a premature tag stop codon , preventing the production of all membrane - bound and soluble isoforms , and therefore it is probably not expressed [ 1 , 152 , 153 ] .
humans bearing allele g*01:05n in homozygosis have been reported [ 154157 ] , a fact that may indicate that soluble hla - g molecules or molecules lacking the 3 domain are sufficient for hla - g function .
the frequency of the g*01:05n allele varies among different populations , ranging from complete absence in amerindian populations from the amazon , mayans from guatemala , and uros from peru [ 139 , 151 , 158 ] , to intermediate frequencies in africa   and higher than 15% in some populations of india , while allele g*01:13n is quite rare [ 152 , 153 ] .
it has been proposed that high g*01:05n frequencies are associated with high pathogen load regions , and intrauterine pathogens would act as selective agents , with increased survival of g*01:05n heterozygous fetuses . in this case , the reduced hla - g1 expression may result in an improved intrauterine defense against infections [ 139 , 151 , 154 , 160 ] . to the best of our knowledge
due to the important role of hla - g in the regulation of the immune response and its relevant function during the course of pregnancy , the overall structure of the molecule has been maintained during the evolution process , preserving major hla - g binding sites to leukocyte receptors and hla - g dimer formation . on the other hand , several variable sites
although a perfunctory analysis of the many variable sites observed in the promoter region of several worldwide populations indicates that some known transcription factor target regions have also been conserved , one can not rule out the influence of the differential action of distinct transcription factors according to promoter region variability .
in contrast , most of the variable sites found in the hla - g 3utr might influence hla - g expression by facilitating or hindering microrna binding and/or influencing mrna stability .
 
 hla - g has a relevant role in immune response regulation . 
 the overall structure of the hla - g coding region has been maintained during the evolution process , in which most of its variable sites are synonymous mutations or coincide with introns , preserving major functional hla - g properties . 
 the hla - g promoter region is different from the classical class i promoters , mainly because ( i ) it lacks regulatory responsive elements for ifn- and nf-b , ( ii ) the proximal promoter region ( within 200 bases from the first translated atg ) does not mediate transactivation by the principal hla class i transactivation mechanisms , and ( iii ) the presence of identified alternative regulatory elements ( heat shock , progesterone and hypoxia - responsive elements ) and unidentified responsive elements for il-10 , glucocorticoids , and other transcription factors is evident . 
 at least three variable sites in the 3 untranslated region have been studied that may influence hla - g expression by modifying mrna stability or microrna binding sites , including the 14-base pair insertion / deletion , + 3142c / g and + 3187a / g polymorphisms . 
 other polymorphic sites have been described , but there are no functional studies on them . 
 the hla - g coding region polymorphisms might influence isoform production and at least two null alleles with premature stop codons have been described . 
 we reviewed the structure of the hla - g promoter region and its implication in transcriptional gene control , the structure of the hla - g 3utr and the major actors of the posttranscriptional gene control , and , finally , the presence of regulatory elements in the coding region . 