the fact that the majority of pre - mrnas are spliced while they are still being transcribed   has led to the proposal that local chromatin structure and histone modifications may play direct roles in regulating splicing .
indeed , several studies have shown that specific histone modifications can affect the association of splicing factors with chromatin and the efficiency of the splicing process [ 2 - 4 ] . a link between nucleosome positioning and exon - intron boundaries
was first proposed in 1991 , long before the functional link between splicing and transcription was established .
recent advances in the computational prediction and experimental verification of nucleosome positioning , based on underlying dna sequence features [ 6 - 8 ] , in combination with recently published high - resolution genome - wide maps of nucleosome positions   and histone modifications   in several organisms , have now enabled genome - wide comparisons of nucleosome positioning , histone modifications , and intron - exon architecture , revealing surprising correlations between these features and throwing light on the question of how chromatin features may help the splicing machinery to distinguish between exons and introns .
recent high - throughput transcriptome analyses have detected alternative splicing in approximately 95% of multi - exon human genes [ 13 - 15 ] and also extensively in mice , plants , flies , and yeast .
alternative splicing is often regulated by trans - acting factors that are differentially expressed in different tissues or metabolic states and bind specific sequence or structural motifs on the pre - mrna , resulting in alternative splicing [ 22 - 24 ] . it has long been a goal of the splicing field to crack the  splicing code : to identify the pre - mrna sequence features that can explain and predict not only the exact sites of constitutive splicing but also the features that determine tissue - specific alternative splicing patterns .
previous work has successfully identified pre - mrna - encoded features that define the precise boundaries of certain classes of constitutively spliced exons [ 26 - 28 ] and additional splicing enhancers and silencers , some of which have been shown to correlate with exon inclusion levels in specific tissues .
however , it has been suggested that pre - mrna - encoded information alone is not sufficient to explain the recognition of short exons in a desert of long introns or the modulation of this process in alternative splicing .
the idea that local chromatin structure may add an additional regulatory layer to splicing , particularly to alternative splicing , is currently gaining momentum , and both correlative evidence and functional evidence are emerging [ 30 - 35 ] . additional insights into the role of rna sequence come from the recent demonstration that by taking hundreds of rna features into account , a 
splicing code based on pre - mrna features is able to qualitatively predict tissue - specific alternative splicing for thousands of vertebrate exons . here , recent advances and current ideas on how chromatin and pre - mrna sequence contribute to constitutive and alternative splicing are reviewed
in 2009 and 2010 , several groups [ 37 - 42 ] used bioinformatic approaches to uncover patterns from published genome - wide maps of nucleosome positioning in human t cells and in caenorhabditis elegans , derived from deep sequencing of micrococcal nuclease - digested chromatin .
these recent bioinformatic analyses [ 37 - 42 ] now show that nucleosomes sit preferentially on exon sequences whereas introns are relatively depleted of nucleosomes ( figure 1 ) .
this remarkable arrangement was observed in several metazoan species and was found to be independent of transcriptional activity , suggesting that the arrangement is an inherent property of chromatin [ 37 - 39,41 ] .
strikingly , the average length observed for metazoan exons ( 140 - 150 base pairs ) corresponds neatly with the length of dna required to wrap a single nucleosome ( 147 base pairs ) .
further correlations to the splicing process were observed by several authors ( e.g. , exons flanked by weak splice sites or by long introns have a higher tendency to be bound by nucleosomes ) , raising the idea that nucleosomes may help the splicing machinery by  marking exons that may otherwise be difficult to recognize ( reviewed in [ 30 - 32 ] ) .
the positions of core rna splicing signals ( red boxes ) , dna sequence properties ( yellow profile ) , and nucleosome occupancy ( blue profile ) on human exon / intron boundaries are drawn approximately to scale according to the distance from the exon end .
core rna splicing signals include the 5 splice site ( ss ) ( position -2 to + 4 ) , which has a variable sequence in higher eukaryotes and a human consensus of agguaag . the branch site ( bs ) has a variable position at -20 to -30 , directly 5 of the polypyrimidine tract ( ppt ) , and a variable sequence , with a human consensus of cuaac .
the ppt ( position -5 to -20 or -30 ) is variable in length ( in humans typically 10 - 25 pyrimidines ) and strongly favors uracil .
the 3 ss ( position -3 to + 1 ) has a variable sequence , and a human consensus of cagg .
each of the core splice signals on the pre - mrna is recognized specifically by a protein or ribonucleoprotein component of the splicing machinery [ 25 - 27 ] .
not shown are the numerous additional splicing regulatory elements , which reside in introns and exons and add specificity to constitutive and tissue - specific splicing .
nucleosome occupancy ( blue profile ) : the average relative nucleosome occupancy across the exon / intron junctions is shown to scale for human activated t cells ( adapted from ) .
the vertical scale represents approximately 1.5-fold higher enrichment on exons compared with introns . note the more profound depletion at the 3 intron end .
the extent of depletion at this site was found to correlate positively with the strength of the ppt .
dna sequence properties ( yellow profile ) : the density of pentamer sequences that disfavor nucleosome binding   was calculated for constitutive exon / intron junctions in   and is drawn approximately to scale .
the maximum value on the vertical scale represents approximately 70% of sequences containing a nucleosome - disfavoring pentamer , and the minimum is approximately 40% .
guanine and cytosine ( gc ) content ( not shown ) may also play a role in nucleosome positioning .
exons have a higher gc content than surrounding intron regions , and this has been proposed to contribute to the observed increased nucleosome occupancy over exons relative to introns . however , there is some disagreement on this point in the literature ( for a discussion , see ) . by analysis of chromatin immunoprecipitation ( chip ) combined with high - throughput sequencing ( chip - seq ) and chip followed by microarray analysis ( chip - chip ) data sets
, several studies have examined correlations between histone modifications and exon - intron boundaries and have reported conflicting results .
several authors conclude that specific histone modifications are enriched on exons compared with introns , suggesting an active marking mechanism , whereas others argue that these apparent enrichments are due mostly to nucleosome positioning , which is independent of modification status .
it has been suggested that these discrepancies may be due mainly to difficulties of normalization of chip data for nucleosome occupancy , compounded by the fact that occupancy studies and modification studies were performed with different techniques ( micrococcal nuclease digestion versus chip ) .
it is also possible that gene - specific differences exist but go undetected in global analyses .
to what extent can we understand the preference of nucleosomes for exonic sequences in terms of known pre - mrna splicing signals ?
figure 1 summarizes the rna , dna , and chromatin features at exon - intron boundaries .
the preference of nucleosomes for exons appears to be a consequence not only of the higher guanine and cytosine content of exons   but also of a high density of sequences that repel nucleosomes   exactly at the intron - exon boundary and a depletion of these sequences within the exon itself relative to the adjacent intron   ( figure 1 ) .
particularly interesting is the polypyrimidine tract ( ppt ) , typically a long ( 10 - 20 nucleotides ) run of uracil bases at the 3 intron end in the pre - mrna , that is specifically recognized by spliceosome components ( figure 1 ) . at the dna level , the ppt corresponds to poly t , one of the strongest nucleosome - repelling sequences identified by kaplan et al . .
thus , it is clear that splicing signal sequences play a role both at the rna level , for recognition by rna - binding proteins of the splicing machinery , and at the dna level , in determining the positions of nucleosomes .
is nucleosome positioning thus merely a coincidence of the rna sequence or does it have a causal role in splicing ?
experimental evidence beyond correlation is currently lacking but several possible roles have been proposed ( summarized in figure 2 ) .
the nascent pre - mrna molecule is shown in black , and the exon is shown as a thicker line .
the large subunit of rna polymerase ii ( pol ii ) is shown in green , transcribing from left to right . the c - terminal domain ( ctd ) of pol ii
dna is shown in blue , with exon - encoding sequences ( thicker lines ) wrapped around nucleosomes ( light blue ) .
red arrows show different points at which the splicing machinery has been shown or proposed to operate .
( 1 ) rna sequences ( shown in figure 1 ) are bound directly by the splicing machinery via protein - rna interactions or ribonucleoprotein - rna interactions .
core signals shown in figure 1 operate on all exons , whereas additional tissue - specific signals act in conjunction with tissue - specific splicing factors to ensure alternative splicing .
( 2 ) the ctd is phosphorylated at different residues upon initiation , elongation , and termination and serves as a binding platform for other proteins .
the ctd may affect pre - mrna processing directly by recruiting splicing factors or indirectly by recruiting nucleosome remodelers and histone modifiers .
( 3 ) when pol ii transcribes through a nucleosome , core histones are transferred behind the transcribing polymerase via a transient dna loop .
unassembled dna or dna in linker sequences presents an opportunity for splicing regulators to bind directly to dna sequences .
( 4a , 4b ) histone tail modifications can recruit regulators of alternative splicing to chromatin .
these may be on reassembled nucleosomes behind pol ii ( 4a ) or on nucleosomes in front of pol ii ( 4b ) .
speed bumps , slowing down the polymerase immediately as it begins transcribing the exon upon which the nucleosome is positioned .
such a mechanism may affect any of steps 1 to 4 by modulating the time available for interactions of trans - acting factors with the exon ( at the dna , rna , or chromatin level ) emerging behind the polymerase .
the models proposed so far fall into one of two non - mutually exclusive classes - the 
recruitment models and the  kinetic coupling models   - and are summarized in figure 2 .
recruitment models favor the idea that nucleosome positioning or nucleosome modifications on exons guide the splicing machinery to the right place ( figure 2 ) , whereas kinetic coupling proposes that the presence of a nucleosome in the path of the polymerase decreases the speed of transcription and thus allows more time for splicing to occur ( figure 2 , arrow 5 ) . in favor of such  speed bump models , single - molecule in vitro experiments
have shown that the speed of rna polymerase ii ( pol ii ) transcription is modulated by nucleosomal barriers .
on the other hand , a recent in vivo study of pol ii transcription rates measured similar speeds on exonic and intronic sequences and showed that although splicing does indeed occur cotranscriptionally , it lags substantially behind the transcription process , being approximately twice as slow .
this raises the question of how much the chromatin features ahead of the polymerase can influence splicing events that take place far behind it ( figure 2 ) .
however , this study was limited to a small number of genes , and it is not known whether they contain exons that are regulated in a chromatin or pol ii elongation - dependent manner ( or both ) . if nucleosome positions do affect splicing , then how could such a system permit the vast amount of flexibility observed in alternative splicing ? several authors have proposed that nucleosome remodelers , a relaxation of nucleosome positioning at alternatively spliced exons , or tissue - specific histone modifications may account for tissue - specific differences in nucleosome positioning or properties and thus facilitate alternative splicing [ 30 - 32,39 ] . in support of this idea , ectopic recruitment of heterochromatin modifications to the vicinity of an alternative exon
was found to reduce pol ii speed and to affect the splicing of that exon .
furthermore , investigation of the fibroblast growth factor receptor 2 ( fgfr2 ) locus revealed cell line - specific enrichment of histone h3 lysine 36 trimethylation ( h3k36me3 ) , which was shown to be required to promote the exclusion of one exon of the gene .
an adaptor protein recruits the proteins required for specific exclusion of this exon to the sites of h3k36 methylation , demonstrating a direct mechanistic link between cell type - specific chromatin modification and exon exclusion .
interestingly , the h3k36me3 enrichments were broadly spread across the fgfr2 locus and were not limited to the excluded exon , suggesting that nucleosome positioning does not play the major role here ; it is the h3k36me3 modification that serves to recruit the necessary factors to the general vicinity of an alternatively spliced exon in the appropriate cell type , although sequence features of the pre - mrna itself determine which exon is to be excluded .
although it remains unclear to what extent alternative nucleosome positions play a role in alternative splicing , a recent study   demonstrates that pre - mrna sequence features are an essential component and perhaps contain sufficient information for many alternative splicing events .
the authors examined the splicing patterns of over 3000 alternative exons in 27 mouse tissues and extracted over 1000 rna sequence features , including known and novel motifs and structural features . from this
the authors compiled 200 features that were diagnostic and qualitatively predictive for tissue - specific alternative splicing .
the predictive power of the code was tested by cross - validation and comparison with experimental data .
depending on the exon type , the code was able to correctly predict alternative splicing in central nervous system and muscle for 65 - 95% of test exons .
importantly , the code is combinatorial , and the authors conclude that large numbers of sequence features are required to ensure tissue - specific splicing .
although this study focuses on the idea that these are pre - mrna sequence features , it is also possible that several of the newly identified features may work at the dna level or at both rna and dna levels as is the case for the ppt   ( figure 1 ) .
in the future , it will be essential to determine the relative contributions of rna sequence features , nucleosome positioning , and histone modifications to constitutive and alternative splicing .
the data so far correlating nucleosome positioning to exon - intron architecture have been limited to few cell types .
it will be important to see whether nucleosome positions are indeed different in different tissues , as has been proposed via thermodynamic competition with tissue - specific transcription factors   and observed for regulatory regions of specific loci .
if this is also the case on a genome - wide scale , then do alternative nucleosome positions correlate with alternatively spliced exons ?
furthermore , it would be extremely interesting to investigate whether any of the rna sequence features that are predictive for alternative splicing   could play an additional role at the dna level ( e.g. , as nucleosome positioning or repelling sequences   or as binding sites for site - specific dna binding proteins that may compete with nucleosomes in a tissue - specific manner ) .
finally , it will be essential to go beyond global correlations and to determine cause and effect for specific loci . to unravel the relative contributions of rna sequence , dna sequence , and chromatin architecture
, it will be essential to overcome the inherent difficulty in such experiments ( i.e. , that any change in dna sequence changes the rna sequence ) .
thus , it will be essential to devise strategies by which nucleosome positions can be modulated without affecting the underlying dna sequence ( e.g. , by manipulating levels of remodelers or competing transcription factors ) .
 recent genome - wide studies have revealed a remarkable correspondence between nucleosome positions and exon - intron boundaries , and several studies have implicated specific histone modifications in regulating alternative splicing . in addition , recent progress in cracking the  splicing code shows that sequence motifs carried on the nascent rna molecule itself are sufficient to accurately predict tissue - specific alternative splicing patterns . 
 together , these studies shed light on the complex interplay between rna sequence , dna sequence , and chromatin properties in regulating splicing . 
Introduction and context
Major recent advances
Future directions
Competing Interests