Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. To express genes, the information present in the DNA blueprint is transcribed by RNA polymerases into RNA molecules. These initial or primary transcripts are often subjected to modifications and processing reactions that mature them into functional molecules. While the processing of some noncoding RNAs such as rRNA and tRNAs are also essential in prokaryotes, extensive processing of protein encoding pre-mRNA transcripts is a unique feature of eukaryotic gene expression.In eukaryotes, 3 major processing reactions are responsible for the maturation of pre-mRNA molecules into mRNAs that are competent for nuclear cytoplasmic export and subsequent translation.While splicing has long been recognized to play an important role in the regulation of gene expression in eukaryotes, the scope for gene regulation at the level of cleavage and polyadenylation has only recently been fully recognized.With the exception of replication dependent histone genes, metazoan protein encoding mRNAs contain a uniform 3′end consisting of a stretch of adenosines. This poly(A) tail is synthesized in a template independent fashion and is the result of an RNA processing reaction, where a multiprotein complex assembles on specific sequences on the pre-mRNAs, called the cleavage and polyadenylation signals . pA signals consist of sequences that flank either side of where the pre-mRNA is endonucleolytically cleaved and subsequently polyadenylated . The corin vivo and in vitro experimental examples. Single nucleotide changes in the A[A/U]UAAA sequence, or its entire removal,non-canonical variants, with a deviation of one or more nucleotides from the A[A/U]UAAA hexamer sequence consensus, represent about 10-20% of all hexamer signals.The importance of the nucleotide composition in the hexamer sequence element is underpinned by numerous In contrast to the hexamer, the DSE has long been considered to be much more tolerant to single nucleotide changes.in vitro based studies revealed that mutations of the dinucleotides at the cleavage site generally resulted in relatively moderate effects on cleavage efficiency. Conversely, a prominent SNP occurring at a 1% frequency in the Caucasian population, altering the cleavage site of the prothrombin gene from CG to CA, significantly increases cleavage efficiency of the pre-mRNA, and has been linked to a higher tendency toward thrombophilia.The point of cleavage occurs between the hexamer and DSE. In mammals, CA and UA appear to be the most frequent dinucleotides that precede the actual site of cleavage,In addition to the core sequence elements, many pA sites possess additional auxiliary sequences that influence their overall strength. These are diverse in location and nucleotide composition, but in most cases feature U/G rich sequences.G-rich auxiliary sequences are some of the best characterized downstream enhancers that may fold into G-quadruplex structures. Several viral and mammalian pA sites have been described where these G-rich sequences significantly influence cleavage efficiency at the pA siteU-rich auxiliary regions are frequently found upstream and downstream of the cleavage site.A UGUA motif has been identified as a USE, which is bound by the cleavage factor I (CFI) complex. UGUA mediated CFIm recruitment to the pre-mRNA can direct cleavage and polyadenylation at non-canonical sites, by anchoring Fip1 and the poly(A) polymerase (PAP) to the pre-mRNA.AUA auxiliary element. A subset of mRNAs are polyadenylated by the non-canonical poly(A) polymerase Star-PAP (see below), which binds an AUA motif in the 3′UTR of its target pre-mRNAs.cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), and mammalian cleavage factors I and II (CFIm and CFIIm). Additional core factors include symplekin, the poly(A) polymerase (PAP), nuclear poly(A) binding protein (PABPN1) and the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). The process of 3′end formation is initiated by the recognition of the respective cis-elements on the nascent transcripts by CPSF and CstF complexes in a cooperative manner.In mammals there are more than 80 proteins associated with the 3′end processing machinery,in vitro.The CPSF protein complex features 6 protein subunits; WDR33, CPSF30, CPSF160, hFip1, CPSF100 and CPSF73, which are required for cleavage of the pre-mRNA. In contrast, only 4 CPSF subunits, CPSF160, CPSF30, WDR33 and hFip1 together with PAP are sufficient to direct AAUAAA dependent poly(A) tail addition (WDR33)WDR33  is a large 146 kDa protein, and despite the essential role its homolog Pfs2p plays in 3′end formation in yeast,in vitro,(CPSF4)CPSF30  is the smallest subunit of the CPSF complex. The involvement of a 30 kDa protein in the recognition of AAUAAA has long been recognized,(CPSF1)CPSF160  was considered to be the major component that mediates the binding of the CPSF complex to AAUAAA. This was primarily based on the observation that a 160 kDa protein can be specifically cross linked to AAUAAA-containing RNA substratesFIP1L1)hFip1  and CPSF73 (CPSF3)CPSF100 , which both contain metallo-β-lactamase and β-CASP domains, form a heterodimer in mammals.CstF is the component of the cleavage and polyadenylation machinery that specifically recognizes the DSE region in pA sites. It is a heterotrimeric protein complex containing the subunits CstF77, CstF64 and CstF50.CSTF2)CstF64 ( mediates the binding to the DSE in the pre-mRNA.in vivo suggests a role for CstF64 in modulating the selection of pA sites during APA.(CSTF3)CstF77  bridges the CstF64 and CstF50 subunits(CSTF1)CstF50 , like CstF77, exists as a homodimerCFIm is a complex is made up of 4 polypeptides: (CPSF5/NUDT21), CFIm68 (CPSF6), CFIm59 (CPSF7)CFIm25  and )CFIm72 PAP ( is a monomer and responsible for the addition of the 3′ polyadenosine tail to a newly synthesized pre-mRNA moleculePABPN1)PABPN1 ( has been shown to contain a single RRM and an arginine rich CTD.Symplekin (SYMPK) is a protein that forms a high-molecular weight complex with CPSF and CstF by interacting with CPSF73, CPSF100 and CstF64. It is suggested to serve as a scaffold for recruiting other factors to the cleavage and polyadenylation complex.HSB pre-mRNA during stress and so safeguard its expression.RBP1)Pol II CTD  factors.While splicing components associated with the 3SS appear to stimulate 3′end formation, the opposite is generally true for splicing components that bind to the 5SS, notably the U1 snRNP. The suppression of pA sites by U1 snRNP has proven to be instrumental for the inhibition of late gene expression in the bovine papilloma type 1 virusSaccharomyces cerevisiaeThe second part of the reaction comprises of the non-templated addition of about 200 adenosines,in vitro systems. Central to the model derived from these observations is that the decoration of the poly(A) tail with PABPN1 provides some means of measuring and controlling the number of adenosines added to the 3′end of the cleaved mRNA. The current model envisages that the growing poly(A) tail coated with PABPN1 folds into a spherical complex that promotes contacts between CPSF, PAP and PABPN1. When the tail extends to 250 adenosines, new PABPN1 proteins can no longer be integrated into the sphere and this leads to the disruption of the interactions between CPSF and PAP. Subsequently PAP remains tethered to the RNA only by its interaction with PABPN1 causing it to readopt the distributive mode and ultimately terminate polyadenylation.in vivo evidence. siRNA mediated depletion of PABPN1 in mouse myoblasts triggered shortening of poly(A) tails and subsequent accumulation of these transcripts in the nucleus.How the precise length of poly(A) tail synthesis is achieved by the polyadenylation complex has been the subject of intensive investigations and much has been learned from reconstituted in vivo, more factors are required. The protein nucleophosmin (NPM1) has been found to associate with many mRNAs only after proper termination of polyadenylation.While CPSF, PAP and PABPN1 are sufficient to direct poly(A) tail length control in reconstituted systems, it appears that The complexity of the pA site architecture combined with the multitude of core and auxiliary interacting factors and interconnection with splicing and transcription provides ample opportunities to regulate pA site recognition. It is therefore perhaps not surprising that alternative pA site usage is prevalent in the mammalian transcriptomes with ∼70% of all human genes subjected to what is commonly known as alternative cleavage and polyadenylation (APA).APA occurs when a single gene has multiple pA sites, compared with constitutive polyadenylation, where one gene has one sole pA site. Like alternative splicing, APA results in multiple distinct RNA transcripts being produced from a single gene. The relative positions of these pA sites in a gene and their usage will ultimately determine the coding and regulatory sequence elements that are present in the different transcript isoforms. Depending on where the different pA sites are located in genes, APA events are separated into 2 major categories: untranslated region APA and coding region APA .Figure Untranslated region APA (UTR-APA) occurs when multiple pA sites are available downstream of the terminal exon of a particular gene. As they are positioned outside the coding regions, differential usage of such pA sites does not alter the coding capacity of the resulting transcript isoforms but instead, alters the lengths of their 3′UTRs. The sequence that is absent in the short isoforms is commonly referred to as the alternative UTR (aUTR), relative to the common UTR (cUTR), which is present in both long and short APA isoforms. The presence or absence of an aUTR may equip a particular mRNA isoform with regulatory elements that influence the fate of the transcript regarding its stability, availability to the translational machinery and subsequent protein output, subcellular localization and even final protein destination. While aUTRs provide scope for potential regulation, it is not always clear what proportion of UTR-APA events have a physiologic impact.Coding region APA (CR-APA) occurs when multiple pA sites are present at positions upstream of the 3′UTR. Thus CR-APA events can produce APA mRNA isoforms that differ in their coding capacity of the final protein product. Cryptic pA sites are also abundant in introns but, as mentioned above, they are actively suppressed by the U1snRNP.In comparison to UTR-APA, it is easier to delineate a physiologic consequence for CR-APA events as they directly impact on the cellular proteome. Despite this, in the literature global analysis of APA is heavily bias toward UTR-APA.http://www.polyasite.unibas.ch]http://genome.bucm.edu.cn/utr/].The sum of all UTR-APA and CR-APA isoforms constitute the cellular APA profile. Advances in next generation sequencing have allowed cellular APA profiles to be analyzed on a transcriptome wide scale and several different pipelines have been developed and are available to investigate APA events . UTR-APAA general pattern of shifts toward the proximal pA sites (UTR shortening) has been seen upon increased cellular proliferation.The activation of neurons by depolarising agents also induces shifts from the distal to proximal pA sites in a subset of genes that are regulated by the MEF2 family of transcription factors.Similar to activation, stressed cells tend to enhance the use of intergenic pA sites and produce 3′extended transcripts.APA profiles have also been found to vary as a function of the differentiation status of cells. This was first described during mouse embryonic development, where the differentiation of C2C12 myoblast cells to myotubes showed a general shift to distal pA sites (UTR lengthening).When specific cell types or tissues are compared, they appear to have characteristic APA profiles.In addition to simple switches between common pA sites, there are several examples of unique pA sites that are only activated in specific tissues such as testisSimilarly, tissue specific pA sites are also used in the brain where they generate specific APA mRNA isoforms with unusually long 3′UTRs ( >10 kb).The conservation of APA sites between species is limited and is restricted to fewer than 500 genes when tandem APA sites are compared between mouse and humans.Saccharomyces cerevisiae, Kluyveromyces lactis, and Debaryomyces hansenii.in vivo as a result of a low-specificity process that has not been subjected to evolutionary optimisation.”Experiments in yeast have compared the conservation of APA between different yeast species including Recently, single cell studies have started to expose the heterogeneity of pA site usage within a population of homogenous stem cells.CCND1.Several studies have observed shifts to usage of proximal pA sites upon cellular transformation and carcinogenesis.How cancer specific APA profiles are established is largely unclear, but the dysregulation of CFIm in some cancer cells can be linked to 3′UTR shortening and provides a mechanistic explanation for this cancer-specific APA feature.In addition to further the understanding of how cancer specific gene expression profiles are established, logging changes in APA profiles from tumor cells may also provide a valuable resource that can be exploited to develop tools to type cancer subtypes,cis-elements in one specific gene that compromises its 3′end processing efficacy. These mutations can result either in loss of function, such as in thalassemia and IPEX syndrome, or gain of function, as exemplified in thrombophilia and mantle cell lymphoma. In the latter, a point mutation in the CCND1 gene causes activation of a novel pA site leading to 3′UTR shortening and transcript and protein overexpression.Many individual APA events have been linked with specific disease phenotypes such as thalassemia and IPEX syndrome.If proteins that regulate cleavage and polyadenylation are mutated, 3′end processing at many different pA sites can be altered and this can lead to changes in APA profiles. For example, the APA profiles between normal and failing hearts differ. While no clear global shifts in 3′UTR lengths are obvious, smaller cohorts of genes in the diseased compared with the normal heart show distinct changes in their APA profiles. Mechanistically the shifts in these APA profiles may be caused by the reduced expression levels of several cleavage and polyadenylation factors, including PABPN1. Interestingly, the levels of functional PABPN1 have before been linked to global shifts from distal to proximal pA sites in cells from oculopharyngeal muscular dystrophy (OPMD) patients.Myotonic dystrophy, a neuromuscular disease, represents an additional example where disease specific APA profiles are dependent on the availability of functional proteins. The Muscleblind-like (MBNL) protein family are key regulators of alternative splicing and mutations that compromise their function are associated with myotonic dystrophy. Mutations that cause loss of function in MBNL proteins also result in large scale bidirectional APA shifts.Extensive APA defects that occur in the cerebellum of amyotrophic lateral sclerosis (ALS) patients have been reported. ALS patients that feature a repeat expansion in the C9orf72, have a higher frequency of short 3′UTR APA isoforms. The cause and the physiologic impact of these APA changes however is unknown.For all these examples it is important to keep in mind that it is not clear whether the shifts in APA profiles are driving disease development or progression, or whether they are a reflection of the disease dependent changes in the cells.As outlined above, APA profiles of cells appear to have characteristic signatures that are dynamic and can change in response to specific cues and disease. To elucidate the physiologic role of global APA changes, it is imperative to unravel the mechanisms and networks that are responsible for shaping these profiles. To do this, a distinction has to be drawn between alterations of APA profiles that are due to specific selection of one pA site over another at the point of cleavage, and changes in APA profiles that are due to post-transcriptional mechanisms, such as degradation of specific isoforms.“active” APA , the distal isoform is translated at a higher rate.The impact of different APA mRNA isoforms on translation efficiency follows a similar pattern. While the APA mediated shortening of UTRs in several mRNAs including While there is not a clear global trend, it is nevertheless clear from these examples that the differential usage of pA sites can alter the stability of selected transcripts and affect their overall protein expression, consequently having a significant impact on major biochemical processes.BDNF,Ranbp1,Impa1,MKK7and KPNB1UTRs can play an important role in directing the subcellular localization of transcriptsAlu repeats in the aUTRs of the Nicolin1 gene is incompatible with nuclear cytoplasmic export in HEK293 cells.APA mediated subcellular localization is not just restricted to highly polarized cells. At the individual gene level, it has been shown that the presence of CD47. The distal APA isoform of CD47 allows binding of HuR and SET resulting in the translated protein to be localized to the plasma membrane. Conversely, the proximal APA isoform translated CD47 locates primarily to the endoplasmic reticulum.UTR-APA controlling localized translation has also been seen with some membrane bound proteins including MAG1I3 as a result of premature polyadenylation at a cryptic pA site located in intron 10 of the gene is present. The shortened MAHI3 protein product promotes mammary cell transformation. However, the activation of this cryptic pA site is unclear as it is not dependent on mutations of local cis-elements and thus is more likely to involve an imbalance of trans-factors.In contrast to UTR-APA, CR-APA by definition, will affect the protein identity, as introducing a pA site upstream of a translational stop codon will result in a protein with a different C-terminus. This may be a simple truncation in the case of exonic CR-APA, or more commonly a different C-terminus in composite-intronic CR-APA or skipped-intronic CR-APA . In the The classic example of physiologic relevant CR-APA is that of the switch between the membrane-bound form of IgM in B-cells to the secreted form in plasma cells.l-prolyl tRNA synthetase that enables the escape of inflammatory genes from the GAIT complex mediated translational silencing.One important constraint of CR-APA isoforms is that they do not result in the introduction of premature translational termination codons. Therefore, they are not subject to nonsense-mediated decay and have the capability of producing alternative protein isoforms with potentially distinct physiologic roles.The advent of high-throughput sequencing technologies has no doubt made a huge contribution to our understanding of 3′end formation and APA. It has allowed us to map pA sites on a transcriptome wide scale and compare their differential usage in tissues and diverse cellular states. While in the past the focus was on mapping “global” trends such as 3′UTR lengthening and shortening in different cellular contexts, in the future the focus has to shift more toward identifying APA events that have a proven physiologic impact. This requires a clear distinction to be made between active and passive APA events. Furthermore, rather than just bioinformatics correlations, the physiologic consequence of such APA events also has to be confirmed by direct experimentation at the individual gene level.The appreciation of APA as a global regulator of gene expression in recent years has also revived a renewed focus on the mechanistic side of pA site recognition. Despite the simplicity of the reaction, it is still unclear how the pA machinery can assemble into functional complexes at pA signals that deviate considerably from the consensus sequences and differ widely in their architecture. Developing methods that can assess the likelihood of a pA site being subjected to regulation


Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as  EPO is produced by specialised cells in the kidney in response to reduced oxygen tension. It binds to the erythropoietin receptor (EpoR) expressed on erythroid progenitor cells to induce proliferation, differentiation, survival and enucleation. Thus, an EPO-EpoR feedback loop re-establishes physiologically appropriate numbers of circulating mature red blood cells in response to need. Exogenous use of EPO, inherited mutations in the EpoR , and acqStat5a and Stat5b, which have 95% amino acid sequence identity . Baithelium . A totalithelium . The 50 de novo motif discovery using MEME [in vitro binding data for STAT5a and STAT5b [We undertook ing MEME  and founing MEME ,33, withing MEME ,26, and d STAT5b ,26,34,35d STAT5b , confirmCish and Socs3 and generic effectors of cell survival such as Bcl2l1  using reported ChIP-seq data ,37. We fs Bcl2l1 . Thus, ws Bcl2l1 . Indeed,s Bcl2l1 .Cish within 10–30 minutes of EPO stimulation [Hprt and β-globin (2>0.95)  or without EPO as a control . Previoumulation ,45, so wβ-globin . We founβ-globin . There w(2>0.95) . Many ofand Pim1 ,26,45,46and Pim1 .There was an overlap between rapidly induced transcripts and nearby ChIP-seq peaks but this was not always the case . In factCish has a robust pSTAT5 ChIP-seq peak at its promoter  which was processed leading to a slower accumulation of mRNA. For example,  60 mins . Thus, tBcl2l1 gene contains a strong pSTAT5 ChIP-seq peak towards the 3′ end of a long second intron; this site overlaps with two GATA1 ChIP-seq peaks  but limited induction of 4sU-labelled RNA by 30 mins (red), so Gpyc was not called as a DEG. However, qRT-PCR and promoter CAGE data clearly show significant induction by EPO , but which had no pSTAT5 ChIP-seq peak within 50kb of the TSS. For example 3.3 fold  with obv3.3 fold , red. We3.3 fold . Howeverhbs1 TSS . The dator Furin . Withoutor Furin . One likWe employed ChIP-seq to determine pSTAT5 occupancy and 4sU-RNA-seq to detect immediate transcriptional targets of EpoR signalling in murine erythroid cells. We found >300 pSTAT5-occupied regions in the erythroid genome. A reduction in the q value threshold from 0.01 to 0.2 only increased peak calls by about 25%, but it also reduced the quality of the peaks , so the true number of pSTAT5-occupied sites is low compared with other erythroid TFs. It is important to remember we undertook the ChIP at one time point after EPO stimulation and in cell lines immortalised at the early erythroblast stage of differentiation. Additional pSTAT5 occupancy may occur at delayed times after EPO stimulation or in more differentiated cells. About 15% of the peaks occur at gene promoters but the majority occur at intronic or intergenic sites. These mostly have features characteristic of enhancers such as bi-directional CAGE tags, the H3K4me1 mark and DNase 1 hypersensitivity.Of the 63 genes identified as EPO immediate early induced, some were unexpected. Manual curation of the encoded proteins places most of them within four functional categories  includinCish and Socs3 ; it has also been reported to be involved in EpoR internalisation [Clint1 (EpsinR) is involved in clathrin-mediated endocytosis (CME) , which ilisation . The N-tRbm25 gene is induced by EPO via binding of pSTAT5 to an intronic enhancer -coding in preference to the Bcl-x(L)-coding mRNA [Rbm25 and thereby splicing of Bcl2l1  is a lysine methyltransferase which induces mono-methylation at histone H4K20. It is essential for nuclear compaction and enucleation . We founcer Figs  and 5C. cer Figs .™ , and 1% Penicillin-Streptomycin . Cells were treated with erythropoietin (EPO) at 10 U/mL prior to harvesting for RNA and ChIP [The J2E cell line  was mainand ChIP  -labelling of newly transcribed RNA with 500 μM 4sU , subsequent isolation, enrichment, and RNA sequencing were performed as recently described .Western blots were incubated with anti-phosphoSTAT5 (1:2000) in 1% BSA overnight. pSTAT5 protein was detected by chemiluminescence using the Pierce ECL Kit  as per the manufacturer’s instructions following incubation with anti-rabbit IgG-HRP   in 1% BSA.ChIP was performed as previously described . We testComputational analysis of raw sequencing data was performed as previously described . HeatmapAll sequencing data generated by this study have been deposited in the Gene Expression Omnibus (GEO) under the accession GSE94301. Supporting DNase I data were from GSE37074, ChIP-seq datasets were accessed from GSE31039 (H3K4me1 and H3K4me3), GSE51338 (GATA1), and additional STAT5 ChIP-seq were from GSE36890, GSE34986, GSE31578 and GSE40930.S1 FigBcl2l1 gene, from mammary epithelia. ChIP was performed on 5 replicate samples following 30 mins EPO induction with the following treatments serving as controls: pSTAT5 Ab (+) or IgG control (−), and treatment with EPO (+) or without (−) for 30 mins. Enrichment of bound DNA was determined by qPCR and expressed as a % of input DNA. (D) ChIP-seq in multiple cell types following 30 mins of EPO induction across the Bcl2l1 gene. Read density profiles for STAT5 across multiple non-erythroid tissues illustrates the basis for ChIP primer design (red tracks). Primers were designed to the previously reported GAS element, and to 1 kb upstream and downstream based upon the binding profiles. Enrichment of pSTAT5 observed in sequencing data (yellow) is consistent with qPCR (C) and prior studies. (E) EPO-induced pSTAT5 occupancy of the Abcg2 gene enhancer. ChIP qPCR was performed as in panel (C), however enrichment was only observed at GAS element 4. (F) Read density profiles of STAT5 ChIP at the Abcg2 gene as described in panel (D). Primers were designed to the previously reported GAS3 and GAS4 elements, and to 1 kb upstream and downstream based upon the binding profiles. Enrichment of pSTAT5 is observed only at GAS4 in sequencing data (yellow), and at a downstream erythroid specific site. (G) CentriMO analysis of discovered MEME motifs i-iii (see (A) Overview of experimental design and results. The J2E murine erythroid cell line was stimulated with EPO (10 U/ml) for the indicated times. An analog of uracil, 4-thiouridine (4sU), was added after 10 minutes to label newly transcribed RNA. 4sU-labelled RNA was isolated after 30 minutes of labelling see  and used-iii see  within p(TIF)Click here for additional data file.S2 Fig(A) Density heat-map of ChIP signal centred on pSTAT5 peaks from J2E cells. The Y-axis represents individual peak regions, and the X-axis represents 10 kb surrounding the summit. Read intensities were normalized to the total number of reads across datasets and hierarchically clustered according to intensity within 500 bp of peak centre. Comparative normalized signals from primary erythroid cells , are shown for DNase I (B), H3K4me3 ChIP (C), and H3K4me1 ChIP (D).(TIF)Click here for additional data file.S3 FigHbb-b1 and Hprt primary transcript by qRT-PCR, using primers targeted to both intron and exons, provide a measure of newly transcribed RNA content. Efficiency of 4sU-labeled RNA isolation from total RNA can be seen by the loss of primary transcript in the eluted fraction . (B) Biological replicates of 4sU-RNA-seq libraries show very high Spearman’s correlation of gene expression r = 0.96 for EPO induced and r = 0.95 for non-induced replicates (C).(A) Comparison of total RNA and isolated 4sU-labeled RNA shows selective enrichment of primary transcripts captured in the 4sU-labeled fraction. Relative levels of (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigPodxl (A-C), Cdk5rap1 (D-F), and Furin (G-I) genes respectively. The overall design and colour coding of tracks is the same as for Podxl TSS which overlap with GATA1 and KLF1 ChIP-seq peaks. However, Podxl is not upregulated by 4sU-RNA-seq (red track) after 30 mins, but shows weak and delayed upregulation of expression which is not significant until 4 hours in qRT-PCR and CAGE. There is a strong pSTAT5 peak at the Cdk5rap1 promoter but no significant upregulation in 4sU-RNA-seq (red track), qRT-PCR or CAGE following EPO induction. Significant upregulation of Furin transcription (red track) can be seen following EPO stimulation. Only a weak pSTAT5 peak can be seen downstream of Furin near to the neighbouring gene, Fes, but there are no other pSTAT5 peaks at the Furin promoter or within 30 kb of the Furin TSS. qRT-PCR for Furin primary pre-spliced transcripts shows dynamic upregulation peaking at 2 hours post EPO stimulation but processed mRNA is not substantially upregulated. CAGE tags at the two alternative Furin promoters show basal expression and delayed gradual upregulation from the second promoter (light green bar) until 4 hours post -EPO stimulation.(A) ChIP-seq, 4sU-RNA-seq, qRT-PCR and CAGE tags from erythroid cells following 30 mins of EPO induction across the (TIF)Click here for additional data file.S6 FigCISH binds activated pY401 in the EpoR via its SH2 domain. It recruits Elongin B via a C-terminal SOCS box and indirectly recruits Elongin C, which then recruits cullin-5 and Rbx1. This complex functions as an E3 ubiquitin ligase, to target EpoR and associated proteins for degradation in the proteasome. SOCS3 functions in a similar way via binding to pY401 but in addition it directly inhibits the kinase activity via competitive displacement of STAT5 binding in the active pocket of JAK2. It achieves this additional function via the N-terminal KIR domain which is not present in CISH.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The induction of these genes is a critical part of the molecular mechanisms underlying successful behavioral adaptation to stress. Our previous work has shown that transcriptional activation of c-Fos and Egr-1 in the hippocampus requires formation of a dual histone mark within their promoter regions, the phosphorylation of serine 10 and acetylation of lysine 9/14 of histone H3. In the present study, using chromatin immuno-precipitation (ChIP), we found that an increase in the formation of H3K9ac-S10p occurs within the c-Fos and Egr-1 promoters after FS stress in vivo and that these histone modifications were located to promoter regions containing cAMP Responsive Elements (CREs), but not in neighboring regions containing only Serum Responsive Elements (SREs). Surprisingly, however, subsequent ChIP analyses showed no changes in the binding of pCREB or CREB-binding protein (CBP) to the CREs after FS. In fact, pCREB binding to the c-Fos and Egr-1 promoters was already highly enriched under baseline conditions and did not increase further after stress. We suggest that constitutive pCREB binding may keep c-Fos and Egr-1 in a poised state for activation. Possibly, the formation of H3K9ac-S10p in the vicinity of CRE sites may participate in unblocking transcriptional elongation through recruitment of additional epigenetic factors.The immediate early genes (IEGs)  This rapid response is the result of a complex cascade of molecular events including intracellular signaling, chromatin modifications and transcription factor binding in the promoter regions of the genes  and ETS-domain containing protein 1 (Elk-1)  whereas a complex of serum response factor (SRF) and pElk-1 (phosphorylated ETS domain-containing protein) binds to serum response elements (SREs) (c-Fos expression (Immediate early genes (IEGs) such as FBJ osteosarcoma oncogene (he genes . Transcr (Elk-1) . Within s (SREs) . A seriepression .CREB is a constitutively expressed protein, which is activated rapidly by phosphorylation at serine 133 (Ser133). Although some studies have found that CREB is constitutively bound to its recognition sites within the genome, the transactivation activity of CREB at those sites  depends upon phosphorylation of CREB at ser133 . The extc-Fos induction (c-Fos (and Egr-1) gene CREs in conjunction with required co-factors, thereby allowing transactivation and gene transcription , CREB is phosphorylated extensively in neurons of the dentate gyrus (DG) in the rat hippocampus , 2002. Induction , epigenecription .in vitro has shown that the induction of c-Fos and Egr-1 requires the formation of phosphorylation and acetylation marks within histone H3 in their gene promoter regions , the hypothesis that formation of phospho-acetylated histone H3 after FS occurs in proximity to CREs within IEG promoters, opening the sites up for pCREB binding, CBP recruitment and transcriptional activation only in the sparse, epigenetically primed neurons. We investigated whether this mark is specific for the CRE region of IEG gene promoters and whether the dual histone mark correlates with increased transcription factor and co-factor binding at these sites. As SRF, in complex with pElk1, can also be involved in initiating IEG transcription , we alsoad libitum. All procedures were approved by the University of Bristol Ethical Committee and by the Home Office of the United Kingdom . All rats were handled for at least 4 days prior to experimentation in order to reduce non-specific handling stress on the day of the experiment.Male Wistar rats  were purchased from Harlan  and group-housed (2–3 per cage). Rats were housed under standard lighting  and environmentally controlled conditions  with food and water available Experiment rats were forced to swim for 15 min in individual glass beakers  filled with water (25°C ± 1) to a depth of ∼21 cm. Subsequently, rats were removed from the beakers, gently towel-dried and returned to their home cages. Baseline groups of rats were left undisturbed in their home cages in the holding room during this time. Rats were killed either at baseline, or at various times after the start of FS (see figure legends for time points). Rats were killed by decapitation following quick (<15 s) anesthesia with isoflurane.For RNA analysis, the brain was removed from the skull and cut into 1 mm coronal slices using a brain matrix. The dorsal DG, the dorsal cornu ammonis (CA regions) and the ventral hippocampal region were then micro-dissected from these slices on a stainless steel box filled with ice using Dumont #7 forceps  under a dissection microscope . DG and CA tissue was snap frozen in tubes in liquid nitrogen and then stored at -80°C. The region micro-dissected spanned the coordinates (anterior-posterior) -2.92 and -3.96 mm from Bregma, according to the atlas of Paxinos and Watson .For ChIP analysis, the brain was removed from the skull and the whole hippocampus was dissected and rapidly snap-frozen in liquid nitrogen before being transferred to -80°C for storage.Unless otherwise stated, all reagents for this study were purchased from Sigma–Aldrich . RNA extraction and analysis was carried out as described previously see ref  using thYwhaz) and hypoxanthine phosphoribosyltransferase 1 (Hprt1) were used as reference genes. See Table 1 for cDNA primer and probe sequences. Relative mRNA expression was calculated using the Pfaffl method (Quantitative polymerase chain reaction (qPCR) was carried out in a StepOnePlus real-time PCR machine (Life Technologies). In each well of a 96 well plate, 2 μl of cDNA was added to 18 μl of reaction solution . Primers and probes were designed using Primer Express software . Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta ]. The pellet was then re-suspended in 1 ml of ice cold lysis buffer  and incubated for 15 min at 4°C.ChIP was essentially conducted as described previously . For eacEach sample was then separated into 200 μl aliquots and sonicated for 3 × 10 cycles . Tubes were gently vortexed in between sets of cycles. After sonication, the recombined samples were centrifuged for 10 min at 14,000 rpm, 4°C and the supernatant (the chromatin) was collected, aliquoted, and snap frozen in dry ice and stored at -80°C.For ChIP, a 200 μl aliquot of chromatin was combined with 1800 μl of dilution buffer  and 10 μl of antibody (Histone H3 acetyl K9 phospho S10 (Abcam), pCREB rabbit polyclonal (Millipore), CBP rabbit polyclonal , CREB-1 rabbit polyclonal [(C-21) X Santa Cruz], pElk-1 mouse monoclonal , the samples were then rotated overnight at 4°C. Each of these antibodies are ChIP grade and have been used successfully for ChIP in the past . ImmunohThe following day, the beads were washed with ice-cold dilution buffer  and the chromatin/ antibody mixture was added to the beads and rotated at 4°C for 3 h. The beads were then collected using a magnetic stand and the unbound liquid was removed. The beads were washed three times with RIPA wash buffer  and twice with 1x Tris-EDTA buffer. The chromatin was then eluted from the beads using 200 μl of elution buffer 1  and 100 μl of elution buffer 2  sequentially, each with 15 min of incubation at room temperature (RT) with regular vortexing. Bound fractions released with each elution buffer were pooled and the crosslinks were reversed by adding NaCl to a final concentration of 200 mM and heating at 65°C overnight. The bound samples were then treated with RNase A  and proteinase K .Input DNA samples were prepared from 20 μl of chromatin, diluted in 280 μl of 1x Tris EDTA buffer (TE) after which NaCl was added to a final concentration of 200 mM and input samples were incubated overnight at 65°C to reverse the DNA-protein crosslinks. The Input samples were then incubated with RNase A  and proteinase K .2O).Input and Bound samples were purified using a QIAquick PCR purification kit  according to the manufacturer’s instructions. DNA from both Input and Bound samples was quantified using a high-sensitivity double-stranded DNA assay kit using a Qubit 2.0 fluorometer (Life Technologies). Bound DNA products and inputs were diluted to 0.05 ng/μl and 0.5 ng/μl respectively in nuclease-free water (nfHTable 2 for primer and probe sequences used after ChIP. Primers were designed to cover CRE and serum responsive element (SRE) sites within the IEG promoters. qPCR was carried out as described for RNA analysis, however, a standard curve was also run, using a serial dilution of rat brain genomic DNA . Enrichment was calculated using the following equation: Bound fraction/ Input fraction (B/I).Levels of target gene DNA within Bound and Input fractions was quantified by qPCR using primers and dual labeled probes modified with FAM as the fluorescent dye and TAMRA as the quencher. Primers and probes were designed using Primer Express software (version 3.0), see YWHAZ and Hprt1. ChIP data is expressed as fold change in enrichment over baseline levels. Graphs present group means ± SEM, sample sizes and individual statistical results are stated within the figure legends. All data were analyzed using One-way ANOVA followed by Dunnet’s or Bonferroni post hoc tests, or by unpaired Student’s t-tests as appropriate. In all cases p < 0.05 was considered to be statistically significant.The statistical and graphical package used to analyze data was GraphPad Prism 6 . Relative RNA expression is presented as a fold-change over baseline levels, after normalization to reference genes, c-Fos and Egr-1 mRNA changes within the DG of the dorsal hippocampus, the CA of the dorsal hippocampus, and the ventral hippocampus were analyzed. Exposure of rats to FS stress caused a highly significant increase in circulating corticosterone levels which peaked in rats killed 30 min after start of the stressor and returned to baseline levels at 180 min after stress. The plasma corticosterone profile from these animals, including relevant methological details, has been published previously . This was unexpected given the previously reported stress-induced increase in pCREB was located predominantly to neurons in the DG . Due to the similarity in results from different hippocampal sub-regions, whole hippocampal samples were processed in subsequent ChIP experiments. We adopted a similar approach in a recent study . Two sets of primers were designed for the c-Fos promoter, one set to cover the SRE and one to cover the CRE. Two sets of primers were also designed for the Egr-1 promoter region, one covering the cluster of SREs and CREs close to the transcriptional start site (named Egr-1 CRE/SRE primers) and one to cover the more distal SREs (named Egr-1 SRE only primers).The rat c-Fos and Egr-1 promoters in hippocampal chromatin after stress. Under baseline conditions there was significant enrichment above input of H3K9ac-S10p  at both the CRE and SRE sites in both the cFos and Egr1 gene promoters. FS increased the formation of H3K9ac-S10p at the CRE site in the proximal promoter region of c-Fos, whilst no stress-induced change in enrichment was seen at the more distal SRE site . Similarly, FS also induced an increase in enrichment of H3K9ac-S10p at the region containing CREs and SREs in the Egr-1 promoter (Egr-1 CRE/SRE primers), whilst no change in enrichment was seen at the more distal SRE only site . A control IgG ChIP was carried out on samples of the same chromatin preparations and no enrichment was found at the CRE sites in either the c-Fos or Egr1 promoter region .FC enhances phospho-acetylation of histone H3 in sparse neurons of the hippocampus and is associated with IEG promoter regions . ChIP wac-Fos and Egr-1 promoters, ChIP studies were carried out to determine the effect of stress on recruitment of CREB and CBP to the CREs in promoter regions of c-Fos and Egr-1 in the hippocampus. Surprisingly, FS did not increase binding of CREB, pCREB, or CBP at the CREs in the c-Fos and Egr-1 promoters at 30 min after stress . Apparently, FS did not raise the level of binding beyond the high level of binding displayed by pCREB and CREB already under baseline conditions. In addition, we investigated pCREB binding at c-Fos and Egr-1 promoters at two earlier time points, 5 and 15 min after stress to rule out any earlier FS-induced changes in binding. As with the 30-min data, there was no change in pCREB binding at CREs in the c-Fos or Egr-1 promoters at these time points after stress . As a positive control, we investigated pCREB binding within a predicted CRE site located at the transcriptional start site of the brain-derived neurotrophic factor (BDNF) transcript IXa. Under baseline conditions, there was no significant enrichment in pCREB binding to the CRE site in Bdnf, however, FS stress caused a significant increase in pCREB binding in this gene . Given this finding was generated using the same purified DNA samples from the pCREB ChIP, it confirms specificity of the high baseline binding and lack of stress-induced increase in pCREB binding at the promoter regions of cFos and Egr1 genes.As FS-induced H3K9ac-S10p formation was found to be associated primarily with CREs rather than SREs in the in vivo .It has been shown that SRF, when bound to SRE sites, subsequently recruits pElk-1, a ternary complex factor, to the site . Furtherin vivo . Therefode novo gene expression within hippocampal neurons. An important step in this process is the induction of the IEGs c-Fos and Egr-1; genes that are expressed rapidly and sparsely within the hippocampus following a stressor  conditions. Despite the lack of an effect of stress on pCREB binding, we did observe a stress-induced increase in IEG mRNA expression throughout the hippocampus indicating other/additional mechanisms may be controlling the transcription of these genes after stress.Successful adaptation in response to a stressful event, such as FS in rats, involves molecular changes and stressor . Inductistressor . Here, wafter FS , surprisc-Fos CRE site in frontal cortex, hippocampus and striatum following electroconvulsive shock stress   . This stte (TPA) . In vivoin vitro .c-Fos or FosB gene transcription either at baseline or 30 min after fear-conditioning  , which has been shown to require histone H3 phospho-acetylation and chromatin remodeling in order to bind to its recognition site within the c-Fos gene promoter   that intpromoter , 2012. Ac-Fos gene induction, it is thought that this gene is constantly poised for activation, however, full transcription of the gene is prevented by a strong block to elongation, close to the transcriptional start site (TSS)  may therefore be an important regulatory mechanism of stimulus-induced transcription  . Proximacription . Releasecription . In factFos gene , providiFos gene  to the gFos gene . Thus, tll types . In vivoongation .c-Fos and Egr-1 gene promoters within the rat hippocampus following FS stress. Furthermore, despite the formation of these marks specifically at CRE sites within these IEG promoters, no increases in CREB, pCREB or CBP binding at these sites were observed after FS stress. Therefore, the original hypothesis that, within the c-Fos and Egr-1 promoters, H3K9ac-S10p is acting as permissive modifications allowing pCREB to bind needs to be rejected. The enhanced formation of H3K9ac-S10p after FS may be involved in the enhanced recruitment of Dnmt3a to the IEG gene promoter/5′-UTR regions. The exact functional significance of H3K9ac-S10p at IEG promoters remains to be determined.In summary, this study has identified that the dual epigenetic mark, H3K9ac-S10p is formed in close proximity to CRE binding sites within Given the clear stress-induced increase in IEG mRNA expression presented here, together with existing immunohistochemical data for the proteins associated with these genes we propose that constitutive binding of pCREB may contribute to maintaining the IEGs in a poised state of activation, which after stress transcends into full transcriptional activation as a result of distinct stress-evoked epigenetic/transcriptional processes at the IEG gene promoters/5′-UTR regions. Further work is required to fully elucidate alternative mechanisms of control, of which proximal-promoter pausing, DNA demethylation events, and/or the involvement of other transcription factors are viable options.SC, KM, and JR designed the study, performed the research, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Regulation of gene expression is achieved by sequence-specific transcriptional regulators, which convey the information that is contained in the sequence of DNA into RNA polymerase activity. This is achieved by the recruitment of transcriptional co-factors. One of the consequences of co-factor recruitment is the control of specific properties of nucleosomes, the basic units of chromatin, and their protein components, the core histones. The main principles are to regulate the position and the characteristics of nucleosomes. The latter includes modulating the composition of core histones and their variants that are integrated into nucleosomes, and the post-translational modification of these histones referred to as histone marks. One of these marks is the methylation of lysine 4 of the core histone H3 (H3K4). While mono-methylation of H3K4 (H3K4me1) is located preferentially at active enhancers, tri-methylation (H3K4me3) is a mark found at open and potentially active promoters. Thus, H3K4 methylation is typically associated with gene transcription. The class 2 lysine methyltransferases (KMTs) are the main enzymes that methylate H3K4. KMT2 enzymes function in complexes that contain a necessary core complex composed of WDR5, RBBP5, ASH2L, and DPY30, the so-called WRAD complex. Here we discuss recent findings that try to elucidate the important question of how KMT2 complexes are recruited to specific sites on chromatin. This is embedded into short overviews of the biological functions of KMT2 complexes and the consequences of H3K4 methylation. N-acetylglucosaminylation, ubiquitinylation, and sumoylation . Thesr review . Thus, Pr review , chromatr review , and ther review . Deregulr review ,10,11. Wr review ,13.One of the best-studied PTMs of core histones is methylation of lysine (K) residues. Various lysines have been demonstrated to be mono-(me1), di-(me2), and/or tri-methylated (me3), a modification that is catalyzed by different lysine methyltransferases (KMTs), referred to as writers. Although methylation of core histones was discovered in 1964 , the firThe molecular analysis of some of the histone lysine methylations has allowed categorizing their functional relevance broadly either in gene activation or in gene repression. For example, tri-methylation of H3K9 and H3K27 are typically associated with gene repression ,20, whilDrosophila, i.e., Su(var)3-9, Enhancer-of-zeste and Trithorax [S-adenosyl-l-methionine (SAM) onto the Nε group of a lysine in substrate proteins [Lysine methylation is catalyzed by KMTs, which contain an evolutionary conserved SET domain, which was named after the first three identified proteins harboring this domain in rithorax . SET domproteins . H3K4 caproteins ,28,29. Kproteins . Additioproteins ,32. Moreproteins . PRDM9  and four different JARID1 proteins (KDM5A-D)  87,88,8,91,92,93Mll1+/− heterozygotes show retarded growth and several additional abnormalities [Hox genes are deregulated in Mll1 and Mll2 knockout cells. Moreover, MLL2 is required in oocytes and during spermatogenesis but not late in embryogenesis and during homeostasis of somatic cells [Knockout studies of different KMT2 family members have demonstrated the importance of these enzymes for mouse development and their functionality in different tissues. Well studied is MLL1 (KMT2A). Its homozygous deletion is embryonically lethal, whereas the malities ,95. Togemalities . Indeed,malities ,99,100. malities . Howeveric cells ,102. Recic cells . This inAsh2l is essential for mouse embryonal development [Ash2l in mouse hepatocytes provokes a disintegration of the liver and subsequent death of the animals [Some subunits of the WRAD complex have also been analyzed by knockout strategies in mice. elopment . The embelopment  and our  animals . A recen animals . Loss of animals . Thus, tMLL1 gene are associated with around 10% of human leukemias [MLL1 translocation may occur as a failure in DNA double strand repair and have been shown to provoke MLL1 fusion to over 80 different partners. Frequently the fusion protein partners are involved, directly or indirectly, in the recruitment of DOT1L (disruptor of telomeric silencing 1-like), a H3K79 dimethyltransferase. H3K79me2 has multiple functions, both in activation and repression of gene transcription, but its role in transformation is not fully understood [MLL1 translocations, which have comparable phenotypes. Nevertheless, the transforming activity of some MLL fusion proteins is still elusive [MLL1 mutations are also linked to the rare Wiedemann-Steiner syndrome [MLL2 contribute to the Kabuki syndrome  and acute myeloid leukemia (AML) [Many of the enzymes that control H3K4 methylation are involved in diseases, most prominently cancer ,106. Theeukemias . MLL1 trderstood . Some fuderstood . The MLL elusive ,111. MLLsyndrome ,113,114.syndrome ,116,117)e . In adde  .A variety of AML and ALL treatment strategies have been developed, which include the inhibition of KMT2 complexes through their interactors or fusion partners . TargetiTogether, the findings summarized above document the importance of KMT2 enzymes and associated factors in embryogenesis and in normal organ development and maintenance. This is further supported by the many genetic alterations associated with the genes encoding these factors that are linked to diseases.Considering the close correlation of H3K4 methylation with open chromatin and gene expression, and of the essential functions of the KMT2 methyltransferase complexes, an important question is how specificity is achieved. In other words, how are KMT2 complexes selectively recruited to distinct sites on chromatin to catalyze gene-specific effects. Moreover, how are individual H3K4 methylation marks, i.e., mono-, di- and tri-methylation, generated at these distinct chromatin sites. H3K4me2/3 marks are positioned near transcription start site (TSS) of genes that are either actively transcribed or are in a state that allows their rapid activation ,137. In Several possibilities can be envisaged regarding how large multi-subunit KMT2 complexes are recruited to specific loci, i.e., in particular to promoters and enhancers. These possibilities include the recruitment by sequence-specific transcription factors (sTFs), specific forms of chromatin that are e.g., defined by certain patterns of histone marks, transcriptional co-factors, basal transcription factors (bTFs), and long non-coding RNAs (lncRNAs) . ConsideArguably the most obvious mechanism is the recruitment of KMT2 complexes by sTFs, similar to many other co-factors that affect chromatin and ultimately control gene expression . This woConsidering the high complexity of the protein composition and the crowded environment in the nucleus, in which the many interactions must be controlled in a time and location-specific manner, locating multi-subunit complexes to specific sites is challenging. The use of different co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) protocols to study the interaction and distribution of factors and histone marks combined with in vitro interaction assays, and gene expression measurements are required to define causality of functionally relevant assemblies of proteins at selective sites in chromatin. Most of the studies that have reported KMT2 recruitments to chromatin do not allow to specify how the enzymes interact with DNA and chromatin, and thus some uncertainty about direct or indirect interaction remains. Also, indirect interactions might involve more than one component bridging KMT2 enzymes with DNA, an aspect that is difficult to analyze. It is worth remembering that protein-protein crosslinks with formaldehyde are less likely than protein-DNA crosslinks because of the proximity of the DNA binding domain of a sTF and the multiple sites on DNA that can be modified by formaldehyde. In contrast, stabilizing protein-protein interactions are limited by the typically limited number of sites that are in sufficient proximity for being crosslinked, which can make it difficult to detect proteins that are indirectly bound to DNA by ChIP .As discussed above, the WRAD core complex appears to be associated with all KMT2 family proteins, which is necessary to assemble functional MTase complexes . In thatWDR5 interacts with multiple sTFs. During the self-renewal of embryonic stem (ES) cells, WDR5 interacts with OCT4 and possibly also with SOX2 and NANOG, suggesting that WDR5 binds to multiple transcription factors . Upon diThe MYC/MAX TF complex also interacts with KMT2 complexes . This coN-methyltransferase-4, aka CARM1) is an arginine-specific MTase that has been demonstrated to regulate myogenic differentiation. It was observed that PRMT4 binds to the sTFs Myogenin and Mef2c and promotes methylation of H3R17 [Myf5 transcription to stimulate myogenic differentiation when methylated by PRMT4. This modification of the N-terminal region of Pax7 results in binding to the C-terminal fragment of KMT2A/B (MLL1/2) [Myf5 promoter.Mef2d is a transcription factor that is involved in murine muscle cell differentiation. Upon p38 MAPK-dependent signaling, the phosphorylation of Mef2d promotes the binding of KMT2 complexes through Ash2l . This stof H3R17 ,147. A m(MLL1/2) . This isHoxc8 as well as genes such as Fgfr3, whose product is important in transcriptional regulation and signal transduction [Additional DNA binding proteins that recruit KMT2 complexes through ASH2L and control H3K4 methylation include NF-Y (nuclear transcription factor Y) , NF-E2  interact through PTIP with Pax2 and Pax5 ,155,156.HOXC6 gene expression and, complementary, depletion of ERα abolished KMT2C/D binding to the HOXC6 promoter [SR-B1 or HOXB9 promoters, respectively [ASH2L locus in some breast cancers appears to result in ASH2L overexpression, which is positively correlated with increased expression of ERα. This is mediated by GATA3, which binds to an enhancer of the gene encoding ERα and recruits ASH2L and a KMT2 complex [The estrogen receptor alpha (ERα) can directly interact with KMT2D/MLL4. The relevant region in KMT2D is the LXXLL motif, which is also present in KMT2C, resulting in H3K4 MTase recruitment . In supppromoter . Yet, otectively ,167. Togectively , can alsectively ,169, as ectively , thus pr complex . The for complex . Recent  complex . It is pThe E2F family of sTFs is closely associated with cell cycle-specific regulation of gene expression, for example by interacting with pocket proteins such as the tumor suppressor RB (retinoblastoma protein) . ThroughAbove we pointed out the transcription factors capable of interacting with KMT2 complexes, which are summarized in RNAP II and its interaction partners are basal transcription factors. Its positioning at the core promoter makes it an important platform to interact with transcriptional co-factors that control local chromatin as well as the chromatin along the transcribed region. WDR82 was found to interact with the C-terminal domain (CTD) of RNAP II and to mediate the recruitment of SET1A/KMT2F complexes . WDR82 iWith the advent of deep sequencing, large numbers of lncRNAs have been identified. The number of genes encoding lncRNAs appears to exceed those encoding proteins. Although typically less abundant than mRNAs, lncRNAs are frequently capped, spliced and poly-adenylated, similar to mRNAs ,188. lncHOTTIP was identified as a regulator of the expression of the HOXA gene cluster [HOTTIP gene is located at the 5′ end of the HOXA cluster and interacts with the genes that are in the 5′ half of the cluster as determined by chromosome conformation capture-carbon copy (5C) analysis, indicating looping and thus close association with the promoters and regulatory sequences of these genes. The knockdown of HOTTIP reduces the expression of these HOXA genes, suggesting that the lncRNA itself is necessary for gene expression. The knockdown also correlates with reduced H3K4me3 in the 5′ half of the HOXA cluster. From these findings it was argued that HOTTIP controls histone modifications. Indeed, HOTTIP lncRNA interacts with the WRAD subunit WDR5 and affects H3K4 methylation [HOX genes [HOTTIP has been found associated with cancer , possibHOXA cluster, also HOXB genes are regulated by a lncRNA and KMT2 complexes. HoxBlinc is encoded by a gene in the HOXB cluster and, similar to HOTTIP, the knockdown or knockout of HoxBlinc results in reduced expression of HOXB genes, accompanied by a decrease in KMT2 complex recruitment and in H3K4me3 [HoxBlinc controls hematopoietic lineage commitment, but beyond this, little is known about the biological function of this lncRNA.In addition to genes of the  H3K4me3 . ThroughFendrr is another lncRNA that interacts with KMT2 complexes [Fendrr was identified as an RNA that is essential for the development of tissues that are derived from lateral mesoderm [Fendrr in tumorigenesis [Fendrr promotes H3K4me3 and/or represses H3K27me3 at some genes. These two histone marks are important to define bivalent chromatin, which is specified by the co-occurrence of the two marks. This is characteristic for poised promoters, which can easily switch from a repressed state to either open or closed chromatin and thus allows quick adjustment to altered gene expression needs, for example in stem and cancer cells [Fendrr interact with KMT2 complexes and with polycomb repressive complex 2 (PRC2) in cells [Fendrr are complex, which is reflected in the antagonistic consequences on PRC2 and KMT2 complex recruitment. As the latter was evaluated using the core component WDR5 as a surrogate of KMT2 complexes, one word of caution is indicated. Both HOTTIP and Fendrr lncRNAs have been shown to interact with WDR5 [omplexes . Fendrr mesoderm . More reigenesis ,206,207.er cells . Consistin cells . Thus, tith WDR5 ,204. As ith WDR5 ,209. MutHOTTIP, HoxBlinc and Fendrr are controlling the activity and the targeting of KMT2 complexes to specific loci is not fully understood. It is possible that these lncRNAs are involved in the local organization of chromatin such as looping [How precisely  looping ,211. By  looping . The lnc looping .HOTTIP and Fendrr bind to WDR5, HoxBlinc interacts with the SET domain of some KMT2 enzymes [While  enzymes . Yeast S enzymes . The del enzymes . MoreoveIn the two sections above, we summarized how sTFs and lncRNAs contribute to the recruitment and positioning of KMT2 complexes. An additional mode can be the direct interaction of these complexes with DNA . It is oAT-hooks and HMG domains were originally discovered in high-mobility group proteins, which are non-histone chromatin proteins. High-mobility group proteins have multiple functions in chromatin organization, in part by competing with histone H1 and by interacting with sTFs, but also by binding to RNA components ,223. HMGIn addition to the catalytic subunits of KMT2 complexes, the WRAD subunit ASH2L has also been shown to directly interact with DNA. The crystal structure of an N-terminal fragment of ASH2L revealed a forkhead-like helix-wing-helix (HWH) domain that binds DNA ,231. MutThe findings summarized in this section suggest that KMT2 complexes can interact directly with DNA. This has implications for co-factor functioning. While most histone modifying enzymes require sTFs for site-selective recruitment, KMT2 complexes may bind directly to DNA, for example CpG islands, and thus affect chromatin at promoters and/or enhancers independent of sTFs. This offers an additional mode for H3K4 methylation at certain loci.The subunits of KMT2 complexes possess domains that suggest binding to chromatin, independent of the above summarized mechanisms that allow sequence-specific binding to certain loci . It is cIt is interesting to note that some KMT2 MTases contain PHD fingers . PHD finIn mixed lineage leukemia, chromosomal translocations between the gene encoding KMT2A/MLL1 and one of more than 80 partner genes have been detected. These translocations result in oncogenic fusion proteins involving the N-terminal one-third of KMT2A . These fThe function of the KMT2C and D PHD fingers are less well explored . A mass KMT2A and B possess bromodomains . Such doWD40 domains are highly conserved structures that have been recognized to be involved in functions associated with chromatin as well as many other activities in cells ,254. TheUbiquitination has also been known to cross-talk with H3K4 methylation. Mono-ubiquitination of several sites in the core histones H2A and H2B have been noted, with H2BK120ub1 being particularly relevant for the discussion here ,264. UbiH3K4 methylation correlates with open, accessible chromatin and polymerase loading. This suggests that these histone marks, mono-, di- and tri-methylation of H3K4, are read by factors that translate the information of these PTMs into polymerase recruitment and activity. Different domains, including Tudor and PHD, have been defined to bind to methylated H3K4 ,246,273.One of the first observations with a clear functional effect was the identification of TFIID as a reader of H3K4me3 . TFIID iBPTF and of WDR5 were phenotypically similar in developing Xenopus embryos, supporting the notion that H3K4me3 is tightly linked to NURF-dependent chromatin remodeling. Moreover, the inhibitor of growth (ING) family of proteins, which possess tumor suppressor functions, sense histone marks [Another H3K4me3 interactor is the nucleosome remodeling factor NURF , an essene marks . The PHDne marks ,284,285)ne marks ,287. PHFne marks ,289. A rne marks .Recent findings suggest a role for H3K4 methylation in mitosis. It is thought that H3K4me3, which is a relatively stable histone mark, is maintained during the whole cell cycle, including mitosis when the chromatin is compacted ,292,293.The studies summarized here suggest that H3K4 methylation is important for regulating gene transcription. H3K4me3 and H3K4me1 correlate well with open chromatin at promoters and enhancers, respectively. Indeed, numerous studies have provided evidence for this, which could not all be mentioned here. Instead, we concentrated exemplarily on some of those processes, for which direct interaction with H3K4 methylated histone H3 was demonstrated. The recent findings indicate that the targeting of the key enzymes that methylate H3K4, the KMT2 or COMPASS complexes, occurs through their recruitment by sTFs, by certain chromatin marks and possibly by lncRNAs. In addition, some evidence for direct binding to DNA has been obtained. Thus, these findings make KMT2 complexes to very broadly recruited key chromatin regulators strongly associated with transcribed genes and their enhancers. Still, relatively few sTFs have been described as recruiters. It will be interesting to see whether sTFs are the main anchors for KMT2 complexes on chromatin or whether other mechanisms, including the combinations of other histone marks, are used to position these complexes onto specific loci. Although some information is available about crosstalk with other histone modifications, certainly much more will have to be learned about the interaction of H3K4 methylation with the many other histone marks that have been identified. Thus, it will be interesting to see the results of future studies that will certainly provide a more complete understanding of the key roles KMT2 complexes and H3K4 methylation play in controlling gene transcription.


The transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting.Our chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II.Our findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression.The online version of this article (doi:10.1186/s12864-017-3589-6) contains supplementary material, which is available to authorized users. The Groucho (Gro)/Transducin-like enhancer of split (TLE) family of corepressors plays crucial roles in the interpretation and integration of multiple spatially and temporally regulated cell intrinsic and extrinsic inputs during metazoan development, thus helping to generate precisely regulated spatial and temporal patterns of gene expression . DrosophGro can repress transcription even when recruited to sites thousands of basepairs away from a core promoter and/or from the activation elements acting on a promoter , 8. WhilDrosophila embryonic development. We find that Gro associates with chromatin in discrete usually transient peaks often clustered upstream of or within regulated genes in a pattern that is not compatible with a simple spreading model for long-range repression. By combining genome-wide chromatin binding and gene expression analysis, we have also identified a set of high-confidence Gro targets, allowing more confident positioning of Gro within the developmentally-regulated gene network. These high confidence targets are highly enriched for promoter-proximal paused RNA polymerase II (Pol II), suggesting a role for Pol II pausing in Gro-mediated repression.To further elucidate the mechanisms and targets of Gro-mediated repression, we have conducted a genome-wide analysis of Gro binding and repression at multiple stages of Gro transgenic flies were described previously ). Embryos were then suspended in homogenization buffer containing 0.3 M (15 ml) sucrose and dounced five times each with loose and tight pestles. Embryo lysate was filtered through 50-micron nylon cell strainer. Clarified lysate was layered over a sucrose cushion consisting of a layer of 1.7 M sucrose (15 ml) underneath a layer of 0.8 M sucrose (15 ml) in homogenization buffer. The samples were centrifuged at 15,000 RCF for 10 min at 4 °C. Pelleted nuclei were resuspended in 250 μl of nuclear lysis buffer . While gently vortexing, an equal volume of NUN buffer  was added drop-by-drop over a period 5 min. Condensed chromatin became visible as a fluffy white precipitate. The solution was then incubated for 20 min on ice and centrifuged at 14,000 rpm for 30 min at 4 °C. The supernatant (primarily nucleoplasm) was discarded and the pellet was resuspended in Trizol reagent (Qiagen). RNA was then purified following the manufacturer’s protocol.Wild-type (OregonR) fly embryos were collected in three successive 2.5 h windows beginning 1.5 h post-deposition. Between 3 and 5 g of embryos were utilized for each fractionation. The chromatin-associated RNA isolation protocol was adapted from previously described procedures , 37. EmbRNA samples were depleted of ribosomal, poly(A)+, and additional RNA contaminants through an affinity depletion procedure adopted from a published protocol . An equirRNA-depleted RNA was concentrated via ethanol precipitation. Size distribution of samples was determined via Agilent 2100 Bioanalyzer (Agilent Technologies). Indexed RNA-seq libraries were generated with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Sequencing was performed on the Illumia HiSeq 2000 sequencing platform . Reads were demultiplexed and mapped as described above for poly(A) + RNA. We obtained at least 13 million reads per replicate . Additional metagene analysis was performed using the ‘metagene’ package of R/Bioconductor [Mean normalized transcript expression levels (FPKM) were generated with DESeq2 (v1.10.0) . Signifionductor .Using a validated affinity purified polyclonal antibody raised against the Gro GP domain , compared to the time point 1  and time point 3 . Gro occupancy is highly dynamic and reversible. Approximately 58% of all Gro binding sites are unique to a single time point, while only about 9% of Gro binding sites are occupied constitutively throughout the time span analyzed Fig. .Fig. 1Grp < 10−10 via Monte-Carlo simulation). These peaks have median widths in the 500–700 bp range , we searched the Gro binding sites at each stage for enriched motifs and then compared the motifs to the binding sites for known Drosophila transcription factors Fig. . Time pozerknullt (zen) and decapentaplegic (dpp) by binding to ventral repression regions (VRRs) in these genes and recruiting Gro [snail (sna) in the ventral region and ventral neuroectodermal genes such as rhomboid (rho) in the ventrolateral region by binding to ventral activation regions (VARs) in these genes [In the early embryo, delineation of the dorsoventral axis requires a maternally-defined concentration gradient of nuclear Dorsal along this axis –60. In vting Gro , 8. In ase genes , 8. The se genes , 61, 62.zen [dpp [As expected, we observe Gro peaks overlapping the known VRRs in the 5’ flanking region of zen  and an izen [dpp  , a VAR in the 5’ flanking region of rho [We also examined VARs in the Dorsal activation targets n of rho –67  [As Gro is known to restrict the expression patterns of many developmental regulators including transcription factors, splicing factors, and signaling molecules  , 2, manyp, etc.) , and simp, etc.) . As Gro We find that the changes in gene expression resulting from Gro overexpression Fig. : S3A areWhile the Gro/TLE family of proteins have traditionally been thought of as obligate corepressors, TLE3, a human Gro family protein, has been shown to serve primarily to activate transcription, although the mechanism remains unknown . HoweverThrough this scoring methodology, we identified 187 potential Gro target genes (which we term “high confidence Gro targets”) across all timepoints  of these sites are only occupied during one of the three time windows we sampled during the first nine hours of embryogenesis and less than 10% are constitutively occupied throughout this period. This dynamic pattern of Gro occupancy likely reflects the shifting availability of sequence-specific transcription factors able to recruit Gro to chromatin.Gro recruits histone deacetylase HDAC1/Rpd3, leading to localized deacetylation of histones and a consequent increase in nucleosome density and repression –17. Deacsog is likely to be a direct Gro target suggests an alternative to the idea that Gro bound to distal sites serves to transfer Gro to promoter proximal regions and that this leads to repression. Chromatin confirmation capture assays suggest that activation of sog may require loop formation between distal regulatory elements and the proximal promoter regions and that repression of sog may involve an anti-looping mechanism [Our finding that echanism . Thus, iOur data show that Groucho binds to many genes that it does not appear to repress. While this could reflect a lack of adequate sensitivity in the expression profiling analysis, we favor the idea that Gro-recruitment is not sufficient for repression. This is suggested by our analysis of Dorsal regulatory targets.zen and dpp, two well-characterized ventrally-repressed genes, consistent with Dorsal-mediated recruitment and repression. Gro occupies the VRRs in these genes, as well as their transcription start sites consistent with the idea that Gro may be delivered from the VRR to the core promoter region, perhaps by a mechanism involving DNA looping.We find that Gro is recruited to the Dorsal-binding VRRs of dpp and tld are among our high confidence Gro targets. This indicates that overexpression of Gro results in reduced dpp and tld expression, an effect that is likely direct. This reduced expression supports the idea that the threshold Dorsal concentration required for repression is sensitive to Gro concentration [The Dorsal repression targets ntration , 23. Thutwi and sog are among our high confidence Gro repression targets, since sufficiently high Gro concentration may stabilize an otherwise unstable co-repressor complex. An alternative possibility that we cannot rule out is that Gro is recruited to VARs by factors other than Dorsal and that this serves to help ensure complete repression of Dorsal activation targets in tissues where they are not normally expressed.Unlike many Gro-dependent repressors , Dorsal is not a dedicated repressor, but also activates expression of certain targets . SurprisPerturbation of Gro activity has severe consequences on the embryonic developmental program . We obseWe note that the approach that we used to identify high confidence targets assumes that the influence of a Gro peak on the expression of a gene tends to decrease as the gene gets further away from the Gro peak. This is a somewhat controversial assumption. For example, a recent analysis showed that long-range looping interactions involving enhancers are very common, with the majority of such interactions occurring at distances of >50 kb . Howeverpannier (pnr), atonal (ato), and patched (ptc). The presence in the network of a hub around pnr, which encodes a transcription factor involved in cardiogenesis [dpp [tolloid (tld) [short gastrulation (sog) [The high confidence Gro targets identified here show that Gro regulates both upstream and downstream elements of a highly-interconnected network of signaling pathways. These include multiple signaling pathways with known Gro involvement, including the Dpp, Wingless, and Epiderimal Growth Factor Receptor (EGFR) signaling pathways , 83. Furogenesis , is of iogenesis –88. Finasis [dpp , tolloidid (tld)  and shoron (sog) , 91, conon (sog) .We find that these high confidence direct targets of Gro-mediated repression are enriched for paused Pol II in the embryo. Consistent with this conclusion, chromatin associated RNA-seq shows that chromatin associated RNA is significantly enriched for transcription start site proximal RNA. These findings suggest that Gro-mediated repression may involve promoter proximal pausing of Pol II.The manner in which Pol II pausing is utilized to regulate transcription remains poorly understood, although multiple non-exclusive mechanisms have been proposed, . One of Given that genes possessing stalled Pol II often continue to be expressed at high levels , 95, PolTo advance our understanding of the mechanisms of development, we have analyzed the patterns of Gro recruitment throughout the Drosophila embryonic genome. We carried out this analysis on Drosophila embryos at multiple stages in the first nine hours of embryogenesis. This is a period when the gene expression profile is rapidly evolving, and we find that the binding of Gro is likewise rapidly changing during this period. The binding sites average three to four nucleosomes in length, and tend to cluster both in distal regions and near the transcriptional start sites of genes. This pattern of binding is inconsistent with one-dimensional spreading of Gro along chromatin and is more supportive of a mode of spreading between cis-regulatory modules and promoters that involves DNA looping. In the future, it may be possible to further test this model using chromatin conformation capture assays in embryos with altered levels of Gro to ascertain the role of Gro in the formation of chromatin loops.Our data also indicate that Gro binding is not the determinative step in Gro-mediated repression as Gro is recruited to both activation and repression targets of the transcription factor Dorsal. We thus suggest that some property of the Gro-containing repressosome  is what determines whether or not repression occurs.We have combined our analysis of Gro binding with measurements of changes in gene expression upon perturbation of Gro levels to identify a set of high confidence targets of Gro repression. Many of these targets comprise a highly interconnected network of genes involved in transcriptional regulation and multiple signaling pathways including the Dpp, Wingless, and EGFR pathways. Analysis of chromatin-associated RNA levels at these target genes reveals that they are enriched for promoter proximal transcripts consistent with a role for Pol II pausing in Gro-mediated repression. Future tests of this model could involve looking at the effect of Gro loss-of-function or overexpression on the extent of Pol II pausing at these high confidence targets using such approaches as Pol II ChIP or GRO-seq.