Drosophila melanogaster implied its function in pyrimidine metabolism. Subsequently, research on Xenopus laevis suggested that it acts as a transcriptional repressor. Finally, studies in humans pointed to a role in pre-mRNA splicing and in mitosis but further research, also in Caenorhabditis elegans and Schizosaccharomyces pombe, demonstrated its much broader activity, namely involvement in the biogenesis of mRNA, and miRNA, piRNA and some other ncRNAs, and in repressive heterochromatin formation. ERH interacts with numerous, mostly taxon-specific proteins, like Mmi1 and Mei2 in S. pombe, PID-3/PICS-1, TOST-1 and PID-1 in C. elegans, and DGCR8, CIZ1, PDIP46/SKAR and SAFB1/2 in humans. There are, however, some common themes in this wide range of processes and partners, such as: (a) ERH homodimerizes to form a scaffold for several complexes involved in the metabolism of nucleic acids, (b) all these RNAs are RNA polymerase II transcripts, (c) pre-mRNAs, whose splicing depends on ERH, are enriched in transcripts of DNA damage response and DNA metabolism genes, and (d) heterochromatin is formed to silence unwanted transcription, e.g., from repetitive elements. Thus, it seems that ERH has been adopted for various pathways that serve to maintain genome integrity.ERH is a 100 to about 110 aa nuclear protein with unique primary and three-dimensional structures that are very conserved from simple eukaryotes to humans, albeit some species have lost its gene, with most higher fungi being a noteworthy example. Initially, studies on  ERH gene which was first reported in the fruit fly Drosophila melanogaster by Tsubota and coworkers in 1994 . The. TheERH ils, see ). The tw others) . The lenERH gene is expressed in all major human adult and fetal normal tissues, which merits its inclusion in the group of housekeeping genes ,32C. ele protein ,41,42. S protein .D. melanogaster, C. elegans and S. pombe show that it localizes primarily to the nucleus [S. pombe, Erh1 also forms nuclear foci in vegetative cells and these foci seem to be the meiotic mRNA degradation sites which, upon entry into meiosis, converge to the single Mei2 dot [S. pombe, C. elegans and humans without nuclear envelope breakdown [Studies on ERH in humans,  nucleus ,44,45,46 nucleus . However nucleus . During  nucleus ,40,42,46Mei2 dot ,37,46. DMei2 dot ,42. Howereakdown .ERH.Since the amino acid sequence of ERH does not deliver any information about the role of ERH in the cell, other means of obtaining such knowledge should be sought. One possible approach is to analyze changes or pathological conditions that accompany mutations and silencing of D. melanogaster, the hypo-morphic mutation in the e(r) gene resulted in the augmentation of aberrations in the wing formation, but in the r mutant background only, and it decreased the level of the r transcript, even in the wild-type r background [e(r) could have some regulatory function in the metabolism of pyrimidines [e(r) null mutants were also morphologically wild-type in the wild-type r background; however, both sexes exhibited decreased viability in addition to diminished fertility in females [r mutant background, these mutations again enhanced the truncated wing phenotype [e(r) null mutation drastically reduced the level of the E(spl) protein, the gene of which is regulated by the Notch signaling pathway [In ckground . It was imidines . The e, delayen stress ,37,46,51observed ,37,46,51Saccharomyces cerevisiae, which does not possess the ERH gene in its genome, the expression of the human ERH resulted in enhanced pseudo-hyphal growth [Interestingly, in l growth .C. elegans erh-2 mutant was sterile, exhibited abnormal chromosome segregation and cell division during embryogenesis, and had decreased levels of mature piRNAs (21U-RNAs) with concomitant accumulation of their precursors [The ecursors ,45.ERH is known in humans. There is one report on the chromosomal deletion of the region with ERH that was associated with mild intellectual disability, congenital heart defects and brachydactyly, among others, in three children, but this haploinsufficiency cannot be attributed exclusively to this gene since the deletion encompassed at least 18 additional protein coding genes [To date, no pathological mutation in ng genes . AccordiERH resulted in the inhibition of cell proliferation in cervical, ovarian, bladder and skin cancer cell lines [KRAS mutation [ERH expression was associated with better survival of colorectal cancer and lung cancer patients with tumors harboring KRAS mutations (though not significantly in the latter patients) [ERH expression had better survival [ERH expression also correlated with better survival of gastric cancer patients, albeit not significantly, and another study showed the opposite in the case of patients with this cancer having an elevated level of the ERH protein [ERH augmented the apoptotic effect of anthocyanins on the gastric cancer cell line [Silencing of ll lines ,19,20,41mutation . The silmutation ,19,54. Imutation ,55. Low atients) . Similarsurvival . Decreas protein ,56. Overell line .ERH resulted in the loss of the mitotic kinesin CENP-E at kinetochores of mitotic chromosomes leading to lack of the proper kinetochore–microtubule attachment and thus to a severe misalignment of chromosomes [CENPE mRNA. Interestingly, this decrease was not the aftermath of less CENPE transcription per se, but resulted from a defect in the splicing of the pre-mRNA [ATR gene that codes for a kinase involved in the response to DNA damage and its repair was affected by the silencing of ERH and resulted in a reduced level of the kinase in cells and its diminished localization to the chromatin following replication stress [ERH led to the deregulation of even more genes in humans. The first pointed to only 87 genes but, within this limited set, revealed an enrichment for genes that, like CENPE, are involved in the cell cycle, as well as for those that, like ATR, are involved in the DNA replication and/or DNA repair [Silencing of omosomes ,53. Thisn stress ,55. Two ERH also up-regulated many human orthologs of the S. pombe meiotic genes and, more generally, derepressed spermatogenic and oogenic genes, as well as other lineage-specific genes, e.g., hepatic ones, and repetitive elements like endogenous retroviruses and the short interspersed nuclear elements (SINEs), but not the long ones (LINEs) [rDNA repeats were unchanged. In accordance with this, the abundance of the histone 3 lysine-9 N-methyltransferase SUV39H1 on heterochromatin was also decreased [Silencing of  (LINEs) . Moreoveecreased .ERH also resulted in changes in post-translational modifications of some nuclear proteins in human cells, namely decreased phosphorylation of the major substrates of the SRPK1 kinase, lamin B receptor that tethers heterochromatin to the nuclear periphery, among others, and the SR splicing factors (SRSFs) [Lastly, it was reported that the silencing of  (SRSFs) ,59,60.ERH expression level observed in cancers and the inhibition of cell proliferation in cancer cell lines by the silencing of ERH. Specifically, these genes could be involved in DNA damage response, as it needs the coordination of the cell cycle and the DNA replication with DNA repair. Moreover, it is conceivable that the observed effects of the e(r) mutations on the expression of the D. melanogaster r gene and the level of the E(spl) protein resulted also from the defect in the splicing of their transcripts. Apart from the decreases in the protein levels in the cell, which are secondary effects, it seems that ERH can probably also indirectly alter the level of phosphorylation of several proteins, some of which play an important role in pre-mRNA splicing, suggesting how ERH could modulate this process. However, ERH appears to be involved in the metabolism not only of mRNA but also other RNA types, like piRNA and some other ncRNAs, as well as in DNA replication, and in the formation of the heterochromatin at protein coding loci and repetitive element loci to repress them.Taken together, these reports suggest that ERH may be necessary for the maintenance of the proper expression levels of the subset of genes that are required for the cell cycle, DNA replication and DNA repair, and that could be achieved by the action of ERH on the pre-mRNA splicing in a positive manner. This is in agreement with the increased Another approach to establishing the role of ERH in the cell is to discover its interactions with partners with known functions in order to obtain a hint of the processes in which ERH could be involved. Although ERH was suggested to be an RNA-binding protein, all of its molecular partners identified so far have turned out to be proteins ,62.Xenopus laevis [Initially, it was ERH that was identified as a binding partner during studies devoted to other proteins. The first of the partners was discovered in the claw frog s laevis . In the s laevis ,65,66. Ss laevis ,66,67. Ns laevis ,69,70. Cs laevis . No proos laevis ,72. Simis laevis . HoweverIn the first attempt, specifically designed to find binding partners of the human ERH, two nuclear proteins, PDIP46/SKAR and CIZ1, were identified by the yeast two-hybrid screen as interacting with it directly ,40. CIZ1+ RNA from nuclear speckles [PDIP46/SKAR  resides in the nucleus, with the exception of the nucleoli and with its highest concentration in nuclear speckles ,42,84. Ispeckles ,95. Intespeckles . The funspeckles .Unfortunately, the significance of the interaction of ERH with either PDIP46/SKAR or CIZ1 has not been studied further. Nevertheless, the binding of the latter and the key ERH amino acid residues for this interaction were also recently confirmed at the structural level (PDB ID: 7X39) . ERH andCENPE gene, among others [ERH was detected as a hit in a large interactome of SMN, a protein that is crucial for the deposition of the hetero-heptamer comprised of the Sm proteins on snRNAs, to form the snRNP core particles in the cytoplasm, which are then imported to the nucleus for the assembly of the spliceosome complex ,99,100. g others . Interesg others ,100,102.g others . Moreoveg others .ATM, a gene coding for another kinase involved in DNA damage repair [From the longest list of the protein partners of the human ERH that has been reported so far, PDIP46/SKAR, CIZ1, CHTOP and PRMT1 were already known . Three ne repair . They are repair ,106. THRe repair . As for e repair ,108. ERHe repair ,109. ERHe repair . Thus, te repair .Direct interactions between the human ERH and SAFB1 or SAFB2, paralogous components of the nuclear matrix, were also reported . SAFB1/2N-methyltransferase SETDB1, the SRC-type kinase activator UNC119, the RNA-binding protein DKFZP564O0523, the GTPase-activating protein DEPDC1B, the uncharacterized protein FAM208B, the mTOR signaling inhibitor DDIT4, the ribosome 40S subunit protein RPS3, the eukaryotic initiation factor 2 subunit EIF2α, the phosphodiesterase SMPDL3A, and the nucleolar zinc-finger protein ZNF330/NOA36 [Other reported partners of the human ERH are the tumor growth inhibitor HOTS, the transcription factor STAT3, the tumor suppressor p53, the Mediator subunit MED31, the transcriptional corepressor TLE1, the heat shock protein HSPA8, the histone 3 lysine-9 30/NOA36 ,119,120.30/NOA36 .D. melanogaster, only two proteins have been found so far to interact with its ERH. One of them is again RPS3 and the other is the ribosome 60S subunit protein RPL19 [D. melanogaster and human ERHs into the nucleus [D. melanogaster ERH was phosphorylated by CK2 in vitro [In in RPL19 . Both of nucleus . The D. in vitro  and the in vitro . Based oin vitro .C. elegans. Instead, ERH-2 binds directly to PID-3/PICS-1 and together they form a stable hetero-tetramer with 2:2 stoichiometry [C. elegans homologs of the mRNA cap-binding protein eIF4E) form a hetero-octamer with 2:2:2:2 stoichiometry named the PETISCO complex, in which TOFU-6 connects PID-3/PICS-1 and IFE-3 [None of the human protein partners of ERH have been reported to interact with any of the two ERH paralogs in hiometry ,35,38,45nd IFE-3 ,35. PETInd IFE-3 ,35. Theind IFE-3 ,35,38. TS. pombe was Mmi1, a foci-forming nuclear protein that promotes the selective elimination of the meiosis-specific transcripts and the down-regulation of Mei2, a master regulator of meiosis, during vegetative growth [rDNA repeats to ensure its integrity [S. pombe ERH (Erh1) could also interact simultaneously with two partners there [sme2 gene, at which locus the dot is formed [The first partner of Erh1 discovered in e growth ,123,124.e growth . Erh1 ane growth ,46.  EMe growth ,128. EMCe growth . EMC cane growth . Erh1 anntegrity . In partntegrity . Interesntegrity . The binrs there ,132. Meis formed ,134,135.Currently, no partner of ERH has been reported in plants.D. melanogaster and, at least in the case of DGCR8, also in C. elegans. Nevertheless, there are some similarities between the identified ERH partners. Many of them are concentrated in various nuclear bodies, e.g., Mmi1, Mei2, CIZ1, PDIP46/SKAR and SAFB1/2 [The small protein has surprisingly many partners, albeit only some of them have been proven to show direct interaction. Particularly, there are many in vertebrates, which are a group with astonishingly conserved ERH, and the plethora of partners can be the very reason for this conservation, i.e., the necessity for the maintenance of interactions with so many partners has kept its amino acid sequence unchanged, as PDIP46/SKAR, CIZ1 and DRGC8 bind to three distinct sites in the human ERH B. Moreov SAFB1/2 ,122,133. SAFB1/2 ,107,114, SAFB1/2 ,106,107, SAFB1/2 ,114,132. SAFB1/2 ,81,114.  SAFB1/2 ,103.6A) and is involved in the formation of the heterochromatin at their loci [N-methyltransferase SETDB1 that was reported to interact with the human ERH, albeit it generates the trimethylated lysine-9 on histone H3 instead of the dimethylated one that was associated with the Erh1 activity in S. pombe [6A residues on Xist and promotes the silencing of the gene transcription on the chromosome X mediated by this RNA [S. cerevisiae, while this yeast lacks MTREC and the ERH gene [Obviously, all these features are not limited to the identified ERH partners. Particularly in mammals, there is the YTH domain-containing protein family with limited similarity to Mmi1 . The fameir loci ,130,138.S. pombe ,116,130.this RNA . Moreovethis RNA ,140. On ERH gene ,140,141.erh-2 in C. elegans and the silencing of ERH in humans led to the similar phenotype, the abnormal chromosome segregation (lagging chromosomes), and the C. elegans TOST-1 might play a role in the splicing [S. pombe and in humans, but in the latter no specific partner has been demonstrated. Thus, we do not yet know all the ERH partners . This applies not only to plants, as mentioned earlier, or to C. elegans, but also to the rest of the above mentioned organisms, and in particular to D. melanogaster, a very convenient model for such studies, in which so far only two partners have been identified experimentally [D. melanogaster proteome and its ERH was functionally replaced by the human ERH [C. elegans uses a unique pathway for their biogenesis [In the fission yeast, ERH is needed for the degradation and the prevention of the nuclear export of the meiotic mRNAs . In the splicing ,45,53. Bmentally . Surprismentally ,143. Moruman ERH . Neverthogenesis ,145. It ERH, while delivering data heading towards its additional functions. ERH seems to be a component of several key complexes involved in nucleic acids’ metabolism, albeit the direct partners that ERH interacts with within some of them are not yet known. In particular, in humans, ERH could be involved not only in pre-mRNA splicing, but also in the initiation, elongation and termination of the transcription (producing pre-mRNA), and in the export of mature mRNAs to the cytoplasm, and these processes are accompanied by the dimethylation of arginine residues. Furthermore, ERH is required for the biogenesis of miRNA.Regardless of whether we already know all the ERH partners, the quite extensive knowledge of the ERH interactome has confirmed the findings from studies with the mutations and the silencing of erh1 in S. pombe is not lethal. The protein is mainly nuclear, but can also be found in the cytoplasm. It has identified neither enzymatic activity nor RNA-binding ability and its only known biochemical action is oligomerization, i.e., its homodimerization that forms the scaffold for the building of the higher-order oligomers. ERH has at least three discrete binding sites for its partners. Surprisingly, the binding to one of these sites, which is near the homodimer interface, does not require the dissociation of the homodimer, and following the binding to the second site, the additional β-strand can be adopted in its loop. ERH could bind two partners simultaneously, e.g., in PETISCO/PICS or in EMC with Mei2, but sequential binding cannot be excluded. There can be competition between ERH and other proteins for binding to ERH partners, so its binding to these partners could liberate the other protein and allow it to execute its function, e.g., the SRPK1 kinase to phosphorylate SR proteins, the S6K kinase to phosphorylate its other substrates, or p21 to bind to the complex of cyclin and cyclin-dependent kinase.ERH is a very small, single domain and globular protein with a unique three-dimensional structure that is tightly packed with the secondary structures, with the exception of one disordered region, the short loop. Its structure, probably invented at the beginning of the evolution of eukaryotes, has stayed exactly the same up to humans, including the size ; however, it is not essential in simple eukaryotes, since in some lineages its gene has been lost and the deletion of S. pombe, C. elegans and humans can be partially explained by the involvement of ERH in the biogenesis of the different types of RNA: mRNA, piRNA, miRNA, and ncRNA, other than the two latter types, in these organisms. However, a common theme regarding all these RNAs is the fact that they all are RNA polymerase II transcripts. In humans, ERH seems to be involved in all stages of mRNA biogenesis in the nucleus, including its export, and particularly in splicing, but only for the subset of mRNAs, mainly of DNA metabolism, chromatin organization and cell cycle genes, along with the processing of the polycistronic transcripts which harbor the suboptimal miRNA hairpins. Likewise, in S. pombe, Erh1 seems to be required for the degradation of the subset of RNAs, namely the meiotic mRNAs and some ncRNAs. Thus, although in humans and S. pombe ERH seems to be a housekeeping gene, ERH is not a general factor but rather an auxiliary one in the biogenesis of some RNA polymerase II transcripts. Interestingly, many stages of the biogenesis of mRNA and its export are accompanied by the dimethylations of arginine residues by PRMT1 and/or PRMT5. It is also worth noticing that, while Erh1 in S. pombe is necessary for the post-transcriptional silencing of the meiotic genes within the cell nucleus and the other ERH-dependent transcripts, like miRNAs, are also involved in the silencing at this level, they do it in the cytoplasm. Lastly, ERH partners in plants have not been identified yet, but one can speculate that, besides those involved in the biogenesis of mRNA, those partners could be some of the proteins required for the biogenesis of siRNA, as this type of RNA is involved in post-transcriptional silencing in plants [S. pombe, i.e., to control meiotic genes, albeit in a modified manner, since Mmi1 seems to be restricted to the fission yeasts.The disparate protein partners in n plants . On the ATR), cell cycle  and chromatin organization genes. On the other hand, the retention of introns is an additional stage in the expression of some genes, which enables the storage of almost fully spliced transcripts in the nucleus, ready for the activation of the removal of the remaining intron(s) by appropriate signals . Such d introns . This do introns . Moreove introns . Thus, i introns ,149,150.rDNA integrity in S. pombe. In C. elegans, to protect the genome integrity in the germline, the piRNA pathway with which ERH is associated blocks the unwanted transcription from dangerous non-self-sequences, such as transposons, by the formation of the repressive heterochromatin. This is also the case in mammals, in which the formation of the repressive heterochromatin in the ERH-dependent manner serves to prevent the untimely expression of the lineage-specific genes, including meiotic and gametogenic ones, and to curb many repetitive elements. However, in S. pombe ERH is required for the assembly of the heterochromatin at some loci only (the heterochromatin islands at the meiotic genes), while the formation of most of the heterochromatin, especially the constitutive one and at retrotransposon loci, depends on a different mechanism that is based on the RNAi pathway with the RITS and RDRP complexes, the latter being absent from metazoans [S. pombe is extended to other lineage-specific genes, while the repression of the repetitive elements is the new one. Moreover, it is possible that ERH could also be involved in the formation of the heterochromatin mediated by chromatin-associated RNAs at transposon loci and during the inactivation of the whole chromosome X [ERH is also involved in the formation of the repressive heterochromatin in order to prevent the untimely expression of the meiotic genes and to protect etazoans ,152. Thumosome X . RegardlMoreover, apart from affecting nuclear DNA replication through the regulation of expression levels and the splicing of some DNA metabolism genes, including DNA replication genes, ERH could also be involved in the assembly of the replication complex and/or in the DNA synthesis and its tethering to the nuclear matrix by its interactions with CIZ1, PDIP46/SKAR and SAFB1/2. However, the action(s) of ERH in the context of these proteins certainly needs more study. Since the activity of ERH seems to be associated with RNA in numerous cases, one can speculate that these processes could also be mediated by chromatin-associated RNAs. In fact, some ncRNAs, like Y RNAs, were implicated in the initiation of the nuclear DNA replication . On the e(r) gene in D. melanogaster manifested only at pyrimidine deficiency, a condition with the dNTP imbalance/lowered dNTP pool that causes DNA replication stress, as noted elsewhere [Lastly, the original mutation in the lsewhere ,55.Taken together, the ERH interactome is a quite extensive network of protein–protein interactions that do not seem to be limited to mRNA splicing and mitosis, as is suggested by the current official ERH name. In fact, these interactions could ensure a much broader role of ERH that consists in the participation in the pathways maintaining genome integrity in a preventive and repair manner, namely by the formation of the repressive heterochromatin, the biogenesis of the transcripts of the subset of genes involved in the DNA damage response and the DNA metabolism, and maybe even by direct interactions with several proteins needed for replication stress and DNA damage responses and DNA repair . Therefo


The PAF1 complex component Rtf1 is an RNA Polymerase II-interacting transcription regulatory protein that promotes transcription elongation and the co-transcriptional monoubiquitination of histone 2B. Rtf1 plays an essential role in the specification of cardiac progenitors from the lateral plate mesoderm during early embryogenesis, but its requirement in mature cardiac cells is unknown. Here, we investigate the importance of Rtf1 in neonatal and adult cardiomyocytes using knockdown and knockout approaches. We demonstrate that loss of Rtf1 activity in neonatal cardiomyocytes disrupts cell morphology and results in a breakdown of sarcomeres. Similarly, Rtf1 ablation in mature cardiomyocytes of the adult mouse heart leads to myofibril disorganization, disrupted cell–cell junctions, fibrosis, and systolic dysfunction. Rtf1 knockout hearts eventually fail and exhibit structural and gene expression defects resembling dilated cardiomyopathy. Intriguingly, we observed that loss of Rtf1 activity causes a rapid change in the expression of key cardiac structural and functional genes in both neonatal and adult cardiomyocytes, suggesting that Rtf1 is continuously required to support expression of the cardiac gene program. Transcription regulation involves the integration of a vast array of inputs, including activators and repressors, chromatin remodelers, histone modification readers and writers, pausing, elongation and termination factors, and other supporting complexes ,2,3,4. TThe PAF1C component Rtf1 is a transcription elongation factor that influences the translocation of RNA Pol II via several highly conserved functional domains. Structural and biochemical examination of the activated transcription complex containing RNA Pol II, DSIF, PAF1C, and Spt6 has revealed that Rtf1’s latch domain extends into a groove in RNA Pol II near the bridge helix and promotes the rate of elongation, suggesting that Rtf1 may directly stimulate translocation by facilitating bridge helix movements . AdditioRtf1 also facilitates co-transcriptional histone modification via a small domain known as the Histone Modification Domain (HMD). The HMD serves as an interaction site for the E2 ubiquitin conjugating enzyme Rad6 and also comes into contact with the nucleosome acidic patch in the cleft between histones 2B and 2A, potentially serving as a bridge between these proteins ,32. In yDrosophila wing morphogenesis [Consistent with Rtf1’s range of transcription regulatory roles, Rtf1 activity influences genes and pathways involved in the embryonic development of multiple tissues. Rtf1 activity negatively regulates erythroid gene expression and the development of the erythroid lineage , but posogenesis ,15. Rtf1ogenesis . Our labogenesis . While tIn this current study, we sought to determine the function of Rtf1 in neonatal and mature cardiac cells. We found that knockdown of Rtf1 in neonatal rat ventricular myocytes (NRVMs) disrupts their morphology, inducing abnormal cellular protrusions and a breakdown of sarcomeres. Similarly, knockout of Rtf1 in the adult mouse myocardium severely compromised cardiac structure and function, resulting in myofibril disorganization, disrupted cell–cell junctions, and fibrosis. Rtf1-deficient hearts fail within three weeks and exhibit a dilated left ventricle and systolic dysfunction. Intriguingly, we found that prior to the onset of functional deterioration, Rtf1-deficient hearts exhibit dysregulation of genes critically involved in calcium ion transport and the contractile machinery of cardiomyocytes. NRVMs likewise displayed changes in the expression of key cardiac structural and functional genes. Together, our data point to a crucial role for Rtf1 in maintaining the cardiac gene program in mature cardiomyocytes.Neonatal rat ventricular myocytes (NRVMs) were isolated from post-natal P1-P3 day old Sprague–Dawley rat pups of mixed gender by UCLA Cardiovascular Research Theme Core services. NRVMs were cultured in DMEM-F12 medium  with 10% horse serum  and 1× Penicillin-Streptomycin (ThermoFisher #15140122) on plates precoated with 0.3% gelatin. Cells were changed into antibiotic-free and serum-free media (with 1% ITS) prior to transfection with siRNAs using Lipofectamine RNAiMAX (ThermoFisher #13778150). A total of 1 million cells were plated per well of a 6-well plate and were transfected with 40 pmol of siRNA. siRNAs used included MISSION siRNA Universal Negative Control #1 (Sigma #SIC001) and Rtf1 siRNA .NRVMs were fixed in 4% paraformaldehyde and permeabilized using acetone. Cells were incubated with α-actinin antibody  overnight at 4 °C. After washing with PBS, sections were incubated with fluorescently labeled secondary antibody and Phalloidin  for 2 h. Cells were mounted using VectaShield fluorescence mounting medium with DAPI (Vector Laboratories) and imaged on a Zeiss LSM 800 confocal microscope.RNA was isolated from mouse heart left ventricle tissue and NRVM cells with Trizol (ThermoFisher) and cleaned up using the Nucleospin RNA kit with on-column DNase digestion (Machery Nagel).cDNA was synthesized with the iScript cDNA Synthesis Kit from Bio-Rad. RT-qPCR was performed using Roche SYBR Green master mix on a Roche LightCycler 480 Real-Time PCR System. qPCR primer sequences are listed in RNA-seq libraries were prepared with the TruSeq RNA Library Prep Kit  and sequenced by the UCLA Technology Center for Genomics & Bioinformatics with an Illumina HiSeq 3000 system. Single-read datasets from mouse left ventricle RNA were mapped to the mm10 genome using Tophat v2.1.0 . Single-tm1a(KOMP)Wtsi, RRID:MMRRC_061682-UCD, was obtained from the Mutant Mouse Resource and Research Center (MMRRC) at the University of California at Davis, an NIH-funded strain repository, and was donated to the MMRRC by The KOMP Repository, University of California, Davis, originating from Pieter de Jong, Kent Lloyd, William Skarnes, Allan Bradley, Wellcome Sanger Institute. This line was crossed with a Flipase expressing strain  to create an Rtf1 conditional knockout allele where exon 3 of Rtf1 is flanked by two loxP sites (Rtf1flox). These mice were crossed into an Myh6:MerCreMer background [flox/flox animals for myocardium-specific knockout experiments. Knockout of Rtf1 was achieved by ad libitum feeding with TAM diet .All mice were maintained in the C57BL/6 background according to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. The mouse strain used for this research project, KOMP ES cell line Rtf1ckground  to produMouse hearts were fixed with 10% neutral buffered formalin and embedded in paraffin, sectioned along the short axis at 5 µm, and stained with hematoxylin and eosin by the UCLA Translational Pathology Core Laboratory. For AFOG staining, 5 µm paraffin sections were prepared by the UCLA Translational Pathology Core Laboratory, deparaffinized, incubated in Bouin’s Fixative for 2.5 h at 56 °C, incubated in 1% phosphomolybdic acid for 15 min, and then stained with AFOG solution  overnight at room temperature.Mouse left ventricle tissue was lysed in RIPA lysis buffer (ThermoFisher #89901) supplemented with 1× Protease and Phosphatase Inhibitors (ThermoFisher #78440). Proteins were subjected to Polyacrylamide Gel Electrophoresis and transferred to a nitrocellulose membrane. Primary antibodies against Rtf1  and H3  were used along with HRP-conjugated secondary antibody . ImageJ was used to perform densitometry comparing the level of Rtf1 protein between samples using histone H3 as the control.Mice were anesthetized with isoflurane and subjected to transthoracic echocardiography with a VisualSonics VEVO 2100 system once every 3–4 days. For each time point, data were acquired from 3–8 mice per condition. Left ventricular end-systolic and diastolic diameters were measured from M-mode echocardiograms of the left ventricle short axis. Ejection fraction and fractional shortening were calculated using the VEVO Lab Cardiac Analysis software package .Short axis cryosections of fresh unfixed mouse hearts were prepared by the UCLA Translational Pathology Core Laboratory. Following thawing and drying of sections, they were fixed with 4% PFA in PBS for 1 min. Sections were then blocked in 10% Goat serum in PBS for 1 h at room temperature, incubated with primary antibodies for 1 h at room temperature, and incubated with secondary antibodies for 30 min at room temperature. Antibodies used included anti-α-actinin , anti-connexin 43 , anti-N-cadherin , anti-mouse IgG AlexaFluor 555 , anti-rat IgG AlexaFluor 488 , and anti-rabbit IgG AlexaFluor 555 .t-test in Microsoft Excel, with a p-value of less than 0.05 considered to be significant. Statistics for differential gene expression were calculated via DESeq2, with an adjusted p-value of less than 0.01 considered to be significant.Data in bar and line graphs are displayed as the mean ± the standard deviation. Statistical analysis for qPCR, Western, and echocardiography data was performed using Student’s To assess whether Rtf1’s transcription regulatory activity is needed for maintaining cardiomyocyte identity after it has been firmly established, we depleted Rtf1 transcripts in neonatal rat ventricular myocytes (NRVMs) using an siRNA knockdown approach. Rtf1 siRNA knockdown was highly effective, resulting in an 83% reduction of Rtf1 transcripts C. IntereCKO) and control animals, respectively , and their hearts exhibited pronounced dilation with thinning of the ventricular walls  promoter . We fed ectively A. Westerar walls B,C.CKO and control hearts after 3 weeks of tamoxifen feeding via immunostaining and histological staining. Both Rtf1CKO and control LVs displayed parallel lines of α-actinin staining, indicating that Z-lines of sarcomeres were intact in Rtf1 knockout hearts. However, myofibrils were more disorganized in Rtf1CKO LVs compared to controls  and Connexin-43 (gap junctions) between myocytes  and red (fibrin). Control myocardium was composed of primarily orange-stained cardiomyocytes with occasional small vessels surrounded by fibrin signal  staining to examine the myocardium and extracellular matrix of Rtf1n signal E. Rtf1CKfibrosis F. TogethCKO and control mice. Prior to feeding with tamoxifen and during the first week of tamoxifen feeding, both Rtf1CKO and control animals exhibited normal left ventricular contractile parameters including ejection fraction (EF), fractional shortening (FS), diastolic volume , and systolic volume   A,C–F. Hofunction C–F and vfunction B. After CKO and control mice at 1, 2, and 3 weeks after initiating tamoxifen feeding and performed RNA-seq and differential expression analysis  for further analysis  were highly enriched in week 1 and 2 Rtf1CKO LVs (CKO animals is partly the result of disrupted mitochondrial function.To explore the biological processes and pathways that were altered in Rtf11CKO LVs D,E, sugg1CKO LVs F, suggesCKO hearts, we found a highly significant enrichment of genes associated with abnormal myocardium morphology (CKO hearts (To further examine the gene expression changes associated with Rtf1 knockout-induced heart failure, we tested the set of genes that was differentially expressed three weeks following the induction of Rtf1 knockout for enrichment of Human Phenotype and KEGG gene set ontologies. Consistent with the structural deterioration we observed in Rtf1rphology A. Intrigrphology B. We selO hearts C. TogethGiven the similar biological processes disrupted at one and two weeks of myocardial Rtf1 knockout, which occur prior to the functional deterioration of the heart after three weeks of knockout, we hypothesized that the associated genes represent a core set of Rtf1-dependent transcriptional regulatory targets. Indeed, we found substantial overlap between the differentially expressed genes at one and two weeks following the initiation of Rtf1 knockout G. A totaCKO LVs, we found that genes critically involved in cardiac biology including ion exchangers/channels, cytoskeleton regulators, transcription factors, and components of cell junctions/ECM, sarcomeres, and signaling pathways were among the “striated muscle”-related downregulated genes in Rtf1 knockdown cells (We also performed RNA-seq to examine the transcriptomic changes underlying the cellular defects we observed in Rtf1-depleted NRVMs. Our differential expression analysis showed that genes involved in catabolism and the microtubule cytoskeleton are highly enriched among those genes upregulated in Rtf1 siRNA-treated cells B, whereawn cells C. OveralEach step in the transcription cycle, from initiation to termination, is subject to regulatory controls that impact the final expression level of a given gene. Rtf1, a member of the evolutionarily conserved PAF1C, regulates transcription at several steps and has been shown to influence a range of developmentally relevant genetic pathways. Our previous finding that Rtf1 is required for cardiac progenitor formation suggested that Rtf1 may be a key regulator of the cardiac gene program . In thisRtf1 knockout hearts are dilated and only very weakly contract, resembling human dilated cardiomyopathy. Rtf1-deficient hearts also exhibit severe defects in intercalated disc structure based on the diminished and diffuse localization of N-cadherin and Connexin-43 proteins C,D. InteNeonatal and adult cardiomyocytes differ in several respects. Maturation of myofibril structure, electrophysiological properties, and metabolism occur in adult cardiomyocytes, and in contrast to neonatal cardiomyocytes, adult cardiomyocytes lack proliferative capacity and exhibit hypertrophic growth . These sCKO myocardium does exhibit myofibril disorganization (Intriguingly, we also observed a difference between the integrity of neonatal and adult sarcomeres upon loss of Rtf1 activity. While the adult Rtf1nization B, Z-linenization I. CardioOur finding that genes relevant to cardiac biology are highly enriched among those genes downregulated upon Rtf1 ablation suggests that Rtf1 positively regulates this set of genes. As an elongation factor, Rtf1 may directly promote the expression of cardiac genes by supporting RNA Pol II processivity. Indeed, diminished RNA Pol II processivity caused by loss of the PAF1C component Paf1 was found to decrease expression of a subset of genes, and strongly impact the transcription of long genes in particular ,58. Rtf1Rtf1 may also promote the expression of cardiac genes by influencing the promoter-proximal pausing of RNA Pol II. During neural crest development, Rtf1 opposes the activity of Cdk9/P-TEFb, a key factor that promotes the transition of RNA Pol II from a paused state to processive elongation, suggesting that Rtf1 promotes promoter-proximal pausing at genes regulating development of the neural crest lineage . SimilarOur study provides the first evidence that Rtf1-dependent transcriptional regulatory activity is continuously required in the myocardial lineage, from embryonic stages to adulthood. Loss of Rtf1 function impacts the cellular structure and gene expression of neonatal cardiomyocytes and the structure and function of the adult heart. Future investigation of the specific transcription regulatory mechanisms influenced by Rtf1 is likely to provide basic information about how cardiac genes are regulated and may lead to therapeutic advances for treating heart disease.


Drosophila has not been investigated and its regulation remains unclear. Here, we performed multiple RNA analyses of a variety sex-specific Drosophila samples and identified over ten thousand circular RNAs, in which hundreds are sex-differentially and -specifically back-spliced. Intriguingly, we found that expression of SXL, an RNA-binding protein encoded by Sex-lethal (Sxl), the master Drosophila sex-determination gene that is only spliced into functional proteins in females, promoted back-splicing of many female-differential circRNAs in the male S2 cells, whereas expression of a SXL mutant (SXLRRM) did not promote those events. Using a monoclonal antibody, we further obtained the transcriptome-wide RNA-binding sites of SXL through PAR-CLIP. After splicing assay of mini-genes with mutations in the SXL-binding sites, we revealed that SXL-binding on flanking exons and introns of pre-mRNAs facilitates back-splicing, whereas SXL-binding on the circRNA exons inhibits back-splicing. This study provides strong evidence that SXL has a regulatory role in back-splicing to generate sex-specific and -differential circRNAs, as well as in the initiation of sex-determination cascade through canonical forward-splicing.Conversely to canonical splicing, back-splicing connects the upstream 3' splice site (SS) with a downstream 5'SS and generates exonic circular RNAs (circRNAs) that are widely identified and have regulatory functions in eukaryotic gene expression. However, sex-specific back-splicing in  Cellular RNA molecules are mostly linear with termini of 5'-phosphate and 3'-hydroxy group; however, circular RNAs have been broadly identified in the past decade, in which free termini are covalently ligated and form a closed loop ,2. Circude novo assembly of circRNAs.In the past decade, biological methods and bioinformatic tools have been developed to identify circular RNAs based on their enrichments by RNase R digestion followed by next generation sequencing ,5,13. RNCis-elements in pre-mRNAs and trans-factors have shown critical roles in the regulation of back-splicing , which allows for the self-regulation of circMbl biogenesis , the master gene in the alternative splicing (AS)-regulated cascade of Drosophila sex-determination pathway. Through PAR-CLIP assay for identification of SXL-binding peaks in the female Kc cells and splicing assay of mutant mini-genes in the male S2 cells, we further demonstrated that SXL can bind to the circularized exon and their flanking sequences for regulation of the female-differentially back-spliced circRNAs.In this study, we first performed multiple RNA-seq including sequencing of the mRNAs, the mRNA and ribosomal RNA-depleted with and without RNase R digestion from a variety of sex-specific WT fly strain used in this study is an isogenic Canton-S strain (Sxl mutant strains f2Sxl  (BDSC 4593)  . Drosophila S2 and Kc were cultured in the complete Schneider's Medium (GIBCO) that contains 10% FBS (GIBCO).The S strain ; the SxlSC 4593)  and Sxl C 58487)  were pur® mRNA Purification Kit (Ambion), and the rest of the RNAs were recovered for further depletion of rRNA (referred to as ‘R-’). In order to completely deplete rRNAs, 6 new probes targeting Drosophila specific 5S and 5.8S rRNAs mixed with previously reported 37 probes  and treated with DNase I (Takara), in which mRNAs (referred to as ‘PolyA’) were isolated using the Dynabeads7 probes  were app7 probes . To obtacutadapt_v1.18, the pair-end reads from the R- and R + samples were then merged using PEAR_v0.9.5, and the merged reads were discarded if < 30nt (Drosophila genome (dm6) using STAR_v2.6.1a, where the parameter ‘-chim_segment_min’ was set to 10 and genes were annotated by a Ensembl_v6.88 database. Transcripts were assembled by cufflinks, and the CIRCexplorer2  of its cognate gene and should be ≥15 in at least one of the samples.To determine a differentially-expressed circRNA between genders/tissues, difference of the BSJ reads between R + samples should be ≥ 4-fold, meanwhile differences of the BSJ reads between R- samples should be at the same direction. Considering low abundance of circRNAs, BSJ reads in R- samples were pre-normalized by the total sequencing reads with reduction of reads from mRNAs and rRNAs using a method derived from actin and βTub60D genes were also listed in Reverse transcription was performed using 1 μg of total RNAs with random primers and RevertAid Reverse Transcriptase (Thermo), and the cDNAs were then amplified by Ex-Taq (TaKaRa). Divergent primers  were desRRM were applied to the Protein G Dynabeads (Invitrogen) that bound with M114 antibody. The co-IPed proteins and RNAs were further analyzed by western blotting and RT-PCR.For western blotting, protein samples were separated on a 10% SDS-PAGE and then transferred to a PVDF membrane . The monoclonal anti-SXL antibody M114 (DSHB), anti-6xHis antibody (H1029 sigma) and anti-α-tubulin antibody T6199 (Sigma) were used for visualization. For co-immunoprecipitation (co-IP), lysates from S2 cells transfected with blank pIZT, pIZT-SXL or pIZT-SXLRRM with six alanine substitutions , TRA, TRA2, DSX-F and DSX-M were cloned into the pIZT vector (Invitrogen). Plasmids with these CDSs were transfected into S2 cells by TransIT Transfection Reagent (Mirus Bio). The WT and mutant mini-genes for generating circRNAs were cloned into the pMT vector, which were then co-transfected with the blank pIZT or pIZT-SXL. Expression of the mini-genes was induced by 0.5 mM CuSO4 at the 12 h post-transfection, and cells were finally collected 48 h later for further isolation of RNAs and proteins (Coding sequences (CDS) of the WT-SXL FBtr0331250) and the mutant SXL and the Sxl was chosen using SnapDragon  with 0.1% SDS, 0.5% deoxycholate and 0.5% NP40]. The cross-linked RNA-protein complexes were co-purified using anti-SXL antibody , which bound to the Protein G Dynabeads. Micrococcal nuclease  was used for digestion of non-protected RNAs, which resulted in protein-bound short RNAs (50–100 nt) on beads. After ligation of 3′-RNA linker and 5′-labeling using γ-32P-ATP, the RNA-protein complexes were separated by SDS-PAGE and transferred to a nitrocellulose membrane, and the desired complexes on the membrane with radioactive signals at the range of 40–60 kDa (SXL is 42 kDa) were cut and digested by proteinase K (Thermo). The RNAs were then recovered by phenol/chloroform extraction and ethanol precipitation, followed by reverse transcription for construction of cDNA libraries and Illumina HiSeq X10 sequencing.PAR-CLIP in ications ,54. BrieCTK_v1.1.3 (cutadapt_v1.18 and extracted by umi_tools (Drosophila genome (dm6) by STAR and annotated by Ensembl_v6.88, in which highly duplicated reads caused by PCR were removed by umi_tools. OmniCLIP was then applied for identification of the SXL-binding peaks  SXLsignal/IgGsignal ≥ 10  and T > C CIMS (FDR ≤ 0.3), or (ii) SXLsignal/IgGsignal ≥ 100  despite T > C CIMS.Raw reads were filtered by K_v1.1.3 , and themi_tools . Clean rng peaks , and CTKdmc_579, the cloned fragment ranges from exons 1 to 5 of the cognate gene crc (CG8669), in which the middle part (3918 nt) of intron 3 without SXL-binding site was removed due to length limitation of cloning, and a trinucleotide (CGA) was inserted into the middle of exon 4 (position 199) to distinguish the exogenous- and endogenous-products. Similar strategies were used for cloning mini-genes that expressing dmc_7247. For construction of mutants of these mini-genes, the multiple Ts in the SXL-binding peaks were substituted by As. For location details and used primers see Figure Mini-genes for expression of circRNAs were constructed into the pIZT vector and transfected into S2 cells. For expression of e crc CG869, in whDrosophila, we sequenced a variety of sex-specific samples, including head, body and gonads from adults of both females and males , sif (ortholog to human TIAM1), para (ortholog to human SCN1A) and slo (ortholog to human KCNMA1) that can generate >30 different circRNAs  than those in the body and ovary respectively; whereas the body and ovary had much less, only 25 and 20 were significantly expressed higher than in the head, respectively Figure , left, avice versa, 21 were stronger in ovary  data Figure  and foundmc_6439 in G5 was specifically back-spliced in ovary but not in testis, while the linear mRNAs from its cognate gene CG12047 were at similar levels in both gonads; and the circRNA dmc_3463 in G6 was specifically back-spliced in testis but not in ovary, while the linear mRNAs from its cognate gene CG40178 were at similar levels  and detected levels of both the circRNAs and linear mRNAs in the sex-specific samples. Except for the two in G2, which only has two circRNAs with fold-changes very close to the cutoff, the other ten circRNAs were confirmed, displaying a pattern of sex-differentially back-spliced activity Figure . For exas Figure .Drosophila is somatic and controlled by a cascade of alternatively spliced genes including Sex-lethal (Sxl), transformer (tra) and doublesex (dsx)  , in whicin males .Drosophila samples, 4 circRNAs  exhibited expression in the female Kc cells but did not in the male S2 cells, whereas the other 4 circRNAs  could not be detected in either Kc or S2 , based on information from a crystal structure of the SXL-RNA complex , whose back-splicing was facilitated by expression of SXL in S2 cells  and constructed their plasmid-borne WT and mutant mini-genes, in which the WT U-rich motifs in the SXL-binding peaks were replaced by A-rich sequences. First, there were two SXL-binding peaks related to the circRNA dmc_579, P1 on its flanking upstream exon and P2 on its back-spliced exon  results in decreased back-splicing, whereas disruption of the SXL-binding on the back-spliced exon results in increased back-splicing.Drosophila samples and found that hundreds of them are sex-differentially and sex-specifically back-spliced. Focusing on the female-specifically back-spliced circRNAs, we demonstrate that the RBP SXL is a key regulator that binds to pre-mRNAs of cognate genes and controls the back-splicing activity in generation of many circRNAs.There are many biochemical layers that could control the production of a cellular circRNA. In this study, we identified thousands of reliable circRNAs from a number of sex-specific msl-2 and Sxl itself, and result in skipped exons of their pre-mRNA splicing , a paralogue of Sxl that is not alternatively spliced between the females and males, has similarities in functions and RNA-binding properties compared to Sxl (In addition, we obtained more than ten thousand RNA-binding sites of SXL transcriptome-wide in the female Kc cells through the PAR-CLIP approach; this will allow for future detail studies on new d to Sxl . In coopAll NGS data have been deposited to NCBI under accession number GSE221102.gkad280_Supplemental_FilesClick here for additional data file.


Here, we produce an analogous chromatin state annotation for mouse based on 901 datasets assaying 14 chromatin marks in 26 cell or tissue types. To characterize each chromatin state, we relate the states to external annotations and compare them to analogously defined human states. We expect the universal chromatin state annotation for mouse to be a useful resource for studying this key model organism’s genome.A large-scale application of the “stacked modeling” approach for chromatin state discovery previously provides a single “universal” chromatin state annotation of the The online version contains supplementary material available at 10.1186/s13059-023-02994-x. Mouse is widely adopted as a model organism for human for many reasons, including its genetic and physiological proximity to humans, relatively short life span, and availability as test subjects for genetic manipulations –3. A weaRecently, we applied an alternative “stacked” modeling approach of ChromHMM to learn chromatin states from over 1000 human datasets representing more than 100 cell types, to generate a universal annotation of the human genome that can be shared across human cell types . This moTo address this, we train a full-stack model with ChromHMM using input data from > 900 mouse datasets of 14 chromatin marks from 26 mouse cell type groups “” sectionWe learned the mouse full-stack model by applying ChromHMM to over 900 mouse epigenomic datasets, similar to how it was previously applied in human , 21 , the latter of which are often marked by signals of both open chromatin mark (ATAC or DNase) and heterochromatin mark H3K9me3 .We defined three groups of states associated with candidate enhancers: active enhancers (mEnhA), weak enhancers (mEnhWk), and transcribed enhancers (mTxEnh). States in the mEnhA group were associated with open chromatin, H3K27ac and H3K4me1. States in the mEnhWk group also showed association with those marks, but at lower levels compared to those in the mEnhA group. States in the mTxEnh group showed signals of open chromatin (ATAC and/or DNase), H3K4me1, H3K27ac, In addition to the mTxEnh group, we defined three additional transcription groups: transcription (mTx), transcription and exons (mTxEx), and weak transcription (mTxWk). States in the mTx group are associated primarily with the transcription marks H3K36me3 and/or H3K79me2/3. Meanwhile, states in transcription and exons group (mTxEx) are associated with both open chromatin and transcription marks. States in the mTxWk group are associated with low levels of transcription marks.We also defined three promoter-associated groups: bivalent promoters (mBivProm), promoter flanking (mPromF), and transcription start sites (mTSS). States in these groups generally had relatively high levels of H3K4me2 and H3K4me3, and for some of them also H3K4me1 and/or open chromatin marks. mBivProm states were also associated with the repressive mark H3K27me3. States in the mTSS group tended to have weaker H3K4me1 levels.Within each group, there were differences among individual states, such as the magnitude of the emission probabilities associated with specific chromatin marks, or their association with different cell type groups Fig. . For exaconstitutive DNase-seq and/or ATAC-seq signal while having relatively limited histone modification signals, had the strongest enrichments with CTCF binding sites in multiple cell types   Figs. .Fig. 2MoAdditionally, we analyzed the enrichment of full-stack states for different chromosomes. This uncovered three states in the polycomb repressed group (mReprPC4-6) that were highly enriched on chromosome X . We found 10 mouse states that are among the top 20 states based on (1) average LECIF score, (2) enrichments for PhastCons element, and (3) enrichments for a specific human full-stack state , following the procedures of . We learned the mouse full-stack chromatin state model for the 901 datasets using the LearnModel command of ChromHMM (v.1.23). We applied the same set of flags as in learning the human full-stack model (-splitrows -holdcolumnorder -pseudo -many -p 6 -n 300 -d -1 -lowmem -gzip), described in Vu and Ernst . We traiconcatenated modeling approach from data of 8 chromatin marks measured in 12 cell type groups at up to 8 distinct stages during mouse fetal development [We obtained per-cell-type 15-chromatin state annotations for 66 reference epigenomes/cell types from Gorkin et al. , with doelopment . We applelopment  to obtaielopment . Second,elopment . These relopment .http://chromosome.sdsc.edu/mouse/download/19-tissues-expr.zip). The provided data contains two gene expression datasets for each tissue type, corresponding to two replicates. We converted the gene expression values for the 19 tissues into We obtained data of gene expression for 19 tissue types in mouse from Ref.  , we calculated the average gene expression of all genes that overlap with the state, while taking into account the genes’ length. We followed the same procedure described in Vu and Ernst . In parteviously :\documenWe then obtained the average gene expression for each full-stack state in each dataset. To calculate the average gene expression for the states in each of the 19 tissue types, we averaged the calculated average expression across the two replicate datasets for the same tissue type.We first lifted over the human full-stack annotation from hg19 to mm10, using the procedure previously described in Vu and Ernst , such thFor each pair of human-mouse states, we determined whether the pair of states correspond to a one-to-one mapping by checking whether the human state was most enriched with the mouse state compared to the other 99 human states and vice versa for the mouse state.Annotations of CpG islands, exons, gene bodies (exons and introns), transcription start sites (TSS), and transcription end sites (TES), 2-kb windows surrounding TSSs (TSS2kb) in mm10 were RefSeq annotations included in ChromHMM (v 1.23) and originally based on annotations obtained from the UCSC genome browser , 31 on JAnnotation of coding gene regions corresponds to coordinates of genes whose feature type is “CDS” from GENCODE mm10 gene annotation, vM25 , accesseAnnotation of assembly gaps in mm10 was obtained from the UCSC genome browser and corresponds to the Gap track , 31, accAnnotations of pseudogenes in mm10 correspond to coordinates of genes whose gene type or transcript type contained “pseudogene” from GENCODE’s mm10 gene annotation, vM25 .Blacklisted regions were downloaded from ENCODE project portal in mm10 from . We noteAnnotations of different repeat classes were downloaded from the UCSC genome browser repeat masker track in mm10, accessed on Jan. 14, 2022 .Annotations of Zinc finger genes in the mouse genome correspond to the coordinates of genes whose name contained “Zfp” based on GENCODE mm10 annotation vM25 .https://hgdownload.gi.ucsc.edu/goldenPath/mm10/bigZips/mm10.chrom.sizes [Annotations of different chromosomes’ coordinates were downloaded from the UCSC genome browser’s data of chromosome sizes in mm10, from om.sizes , 31.https://github.com/ernstlab/LECIF [LECIF scores measure the human-mouse evidence of conservation at the functional genomics level and were downloaded in version 1.1 from ab/LECIF . For eacCTCF peaks data for mouse were downloaded as .bed files format from the ENCODE portal , 34–36. https://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/phastConsElements60way.txt.gz).PhastCons conserved elements  based onThe sources for external annotations for enrichment analyses are as follows (all download links are listed in Additional file Additional file 1: Supplementary Figure 1. Mouse full-stack states transition probabilities. Supplementary Figure 2. Positional enrichments of full-stack states around annotated transcription start sites and transcription end sites. Supplementary Figure 3. Mouse full-stack states enrichments with different chromosomes. Supplementary Figure 4. Mouse full-stack states enrichments with different classes of repeats. Supplementary Figure 5. Enrichment of select mouse full-stack states with different classes of repeat elements. Supplementary Figure 6. Full-stack states maximum-enrichments with annotated concatenated-model chromatin states in 66 mouse reference epigenomes. Supplementary Figure 7. Estimated probabilities of per-cell-type concatenated-model chromatin states overlapping with mouse full-stack states. Supplementary Figure 8. Enrichments of mouse full-stack states with human full-stack states. Supplementary Figure 9. Mouse full-stack states’ relationship with LECIF scores, human full-stack states and phastCons elements. Supplementary Figure 10. Analysis for the number of states.Additional file 2. Metadata and download links for input data used for model learning, CTCF elements.Additional file 3. Summary characterizations of mouse full-stack states.Additional file 4. Mouse full-stack states’ average LECIF scores, enrichments with human full-stack states, repeat classes, chromosomes and CTCF elements.Additional file 5. Mouse full-stack states’ relationships with per-cell-type annotations.Additional file 6. Review history.