PHF2-mediated H3K9me balance orchestrates heterochromatin stability and neural progenitor proliferation.

Heterochromatin stability is crucial for progenitor proliferation during early neurogenesis. It relays on the maintenance of local hubs of H3K9me. However, understanding the formation of efficient localized levels of H3K9me remains limited. To address this question, we used neural stem cells to analyze the function of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass-spectroscopy and genome-wide assays, we show that PHF2 interacts with heterochromatin components and is enriched at pericentromeric heterochromatin (PcH) boundaries where it maintains transcriptional activity. This binding is essential for silencing the satellite repeats, preventing DNA damage and genome instability. PHF2's depletion increases the transcription of heterochromatic repeats, accompanied by a decrease in H3K9me3 levels and alterations in PcH organization. We further show that PHF2's PHD and catalytic domains are crucial for maintaining PcH stability, thereby safeguarding genome integrity. These results highlight the multifaceted nature of PHF2's functions in maintaining heterochromatin stability and regulating gene expression during neural development. Our study unravels the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis.

Imaging analysis of six human histone H1 variants reveals universal enrichment of H1.2, H1.3, and H1.5 at the nuclear periphery and nucleolar H1X presence.

Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.

Genomic profiling of six human somatic histone H1 variants denotes that H1X accumulates at recently incorporated transposable elements.

Histone H1, a vital component in chromatin structure, binds to linker DNA and regulates nuclear processes. We have investigated the distribution of histone H1 variants in a breast cancer cell line using ChIP-Seq. Two major groups of variants are identified: H1.2, H1.3, H1.5 and H1.0 are abundant in low GC regions (B compartment), while H1.4 and H1X preferentially localize in high GC regions (A compartment). Examining their abundance within transposable elements (TEs) reveals that H1X and H1.4 are enriched in recently-incorporated TEs (SVA and SINE-Alu), while H1.0/H1.2/H1.3/H1.5 are more abundant in older elements. Notably, H1X is particularly enriched in SVA families, while H1.4 shows the highest abundance in young AluY elements. Although low GC variants are generally enriched in LINE, LTR and DNA repeats, H1X and H1.4 are also abundant in a subset of recent LINE-L1 and LTR repeats. H1X enrichment at SVA and Alu is consistent across multiple cell lines. Further, H1X depletion leads to TE derepression, suggesting its role in maintaining TE repression. Overall, this study provides novel insights into the differential distribution of histone H1 variants among repetitive elements, highlighting the potential involvement of H1X in repressing TEs recently incorporated within the human genome.

Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation.

Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to the high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA-processing machineries have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors, including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. H1s are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation of RNA polymerase II (RNAPII). Thus, H1 variants determine splicing fate by modulating RNAPII elongation.

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner.

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.

Nucleosome destabilization by polyamines.

The roles and molecular interactions of polyamines (PAs) in the nucleus are not fully understood. Here their effect on nucleosome stability, a key regulatory factor in eukaryotic gene control, is reported, as measured in agarose embedded nuclei of H2B-GFP expressor HeLa cells. Nucleosome stability was assessed by quantitative microscopy [1,2] in situ, in close to native state of chromatin, preserving the nucleosome constrained topology of the genomic DNA. A robust destabilizing effect was observed in the millimolar concentration range in the case of spermine, spermidine as well as putrescine, which was strongly pH and salt concentration-dependent, and remained significant also at neutral pH. The integrity of genomic DNA was not affected by PA treatment, excluding DNA break-elicited topological relaxation as a factor in destabilization. The binding of PAs to DNA was demonstrated by the displacement of ethidium bromide, both from deproteinized nuclear halos and from plasmid DNA. The possibility that DNA methylation patterns may be influenced by PA levels is contemplated in the context of gene expression and DNA methylation correlations identified in the NCI-60 panel-based CellMiner database: methylated loci in subsets of high-ODC1 cell lines and the dependence of PER3 DNA methylation on PA metabolism.

Coordinated changes in gene expression, H1 variant distribution and genome 3D conformation in response to H1 depletion.

Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1.2/H1.4 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local GC content and that their distribution is robust with respect to H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of topologically associating domains (TADs). Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells.

TADs enriched in histone H1.2 strongly overlap with the B compartment, inaccessible chromatin, and AT-rich Giemsa bands.

Giemsa staining of metaphase chromosomes results in a characteristic banding useful for identification of chromosomes and its alterations. We have investigated in silico whether Giemsa bands (G bands) correlate with epigenetic and topological features of the interphase genome. Staining of G-positive bands decreases with GC content; nonetheless, G-negative bands are GC heterogeneous. High GC bands are enriched in active histone marks, RNA polymerase II, and SINEs and associate with gene richness, gene expression, and early replication. Low GC bands are enriched in repressive marks, lamina-associated domains, and LINEs. Histone H1 variants distribute heterogeneously among G bands: H1X is enriched at high GC bands and H1.2 is abundant at low GC, compacted bands. According to epigenetic features and H1 content, G bands can be organized in clusters useful to compartmentalize the genome. Indeed, we have obtained Hi-C chromosome interaction maps and compared topologically associating domains (TADs) and A/B compartments to G banding. TADs with high H1.2/H1X ratio strongly overlap with B compartment, late replicating, and inaccessible chromatin and low GC bands. We propose that GC content is a strong driver of chromatin compaction and 3D genome organization, that Giemsa staining recapitulates this organization denoted by high-throughput techniques, and that H1 variants distribute at distinct chromatin domains. DATABASES: Hi-C data on T47D breast cancer cells have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE147627.

Towards understanding the Regulation of Histone H1 Somatic Subtypes with OMICs.

Histone H1 is involved in the regulation of chromatin higher-order structure and compaction. In humans, histone H1 is a multigene family with seven subtypes differentially expressed in somatic cells. Which are the regulatory mechanisms that determine the variability of the H1 complement is a long-standing biological question regarding histone H1. We have used a new approach based on the integration of OMICs data to address this issue. We have examined the 3D-chromatin structure, the binding of transcription factors (TFs), and the expression of somatic H1 genes in human cell lines, using data from public repositories, such as ENCODE. Analysis of Hi-C, ChIP-seq, and RNA-seq data, have revealed that transcriptional control has a greater impact on H1 regulation than previously thought. Somatic H1 genes located in topologically associated domains (TADs) show higher expression than in boundary regions. H1 genes are targeted by a variable number of transcription factors including cell cycle-related TFs, and tissue-specific TFs, suggesting a fine-tuned, subtype-specific transcriptional control. We describe, for the first time, that all H1 somatic subtypes are under transcriptional co-regulation. The replication-independent subtypes, which are encoded in different chromosomes isolated from other histone genes, are also co-regulated with the rest of the somatic H1 genes, indicating that transcriptional co-regulation extends beyond the histone cluster. Transcriptional control and transcriptional co-regulation explain, at least in part, the variability of H1 complement, the fluctuations of H1 subtypes during development, and also the compensatory effects observed, in model systems, after perturbation of one or more H1 subtypes.

HIV "shock and kill" therapy: In need of revision.

The implementation of antiretroviral therapy 23 years ago has rendered HIV infection clinically manageable. However, the disease remains incurable, since it establishes latent proviral reservoirs, which in turn can stochastically begin reproducing viral particles throughout the patient's lifetime. Viral latency itself depends in large part on the silencing environment of the infected host cell, which can be chemically manipulated. "Shock and kill" therapy intends to reverse proviral quiescence by inducing transcription with pharmaceuticals and allowing a combination of antiretroviral therapy, host immune clearance and HIV-cytolysis to remove latently infected cells, leading to a complete cure. Over 160 compounds functioning as latency-reversing agents (LRAs) have been identified to date, but none of the candidates has yet led to a promising functional cure. Furthermore, fundamental bioinformatic and clinical research from the past decade has highlighted the complexity and highly heterogeneous nature of the proviral reservoirs, shedding doubt on the "shock and kill" concept. Alternative therapies such as the HIV transcription-inhibiting "block and lock" strategy are therefore being considered. In this review we describe the variety of existing classes of LRAs, discuss their current drawbacks and highlight the potential for combinatorial "shocktail" therapies for potent proviral reactivation. We also suggest investigating LRAs with lesser-known mechanisms of action, and examine the feasibility of "block and lock" therapy.

BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells.

Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process modulated by coactivator and corepressor proteins. Breast Cancer Amplified Sequence 2 (BCAS2), is a poorly studied ERα coactivator. In this work, we characterize some of the mechanisms through which this protein increases ERα activity and how this promotes carcinogenic processes in breast cancer cells. Using protein-protein interaction and luciferase assays we show that BCAS2 interacts with ERα both in vitro and in vivo and upregulates transcriptional activation of ERα directly through its N-terminal region (AF-1) and indirectly through its C-terminal (AF-2) region, acting in concert with AF-2 interacting coactivators. Elevated expression of BCAS2 positively affects proliferation, clonogenicity and migration of breast cancer cells and directly activates ERα regulated genes which have been shown to play a role in tumor growth and progression. Finally, we used signal transduction pathway inhibitors to elucidate how BCAS2 is regulated in these cells and observed that BCAS2 is preferentially regulated by the PI3K/AKT signaling pathway. BCAS2 is an AF-1 coactivator of ERα whose overexpression promotes carcinogenic processes, suggesting an important role in the development of estrogen-receptor positive breast cancer.

Epigenetic mechanisms in health and disease: BCEC 2017.

The Barcelona Conference on Epigenetics and Cancer (BCEC) entitled "Epigenetic Mechanisms in Health and Disease" was held in Barcelona, October 26-26, 2017. The 2017 BCEC was the fifth and last edition of a series of annual conferences organized as a joint effort of five leading Barcelona research institutes together with B-Debate. This edition was organized by Albert Jordan from the Molecular Biology Institute of Barcelona (IBMB-CSIC) and Marcus Bushbeck from the Josep Carreras Leukaemia Research Institute (IJC). Jordi Bernués, Marian Martínez-Balbás, and Ferran Azorín were also part of the scientific committee. In 22 talks and 51 posters, researchers presented their latest results in the fields of histone variants, epigenetic regulation, and chromatin 3D organization to an audience of around 250 participants from 16 countries. This year, a broad number of talks focused on the epigenetic causes and possible related treatments of complex diseases such as cancer. Participants at the 2017 BCEC elegantly closed the series, discussing progress made in the field of epigenetics and highlighting its role in human health and disease.

A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other "Shock" Drugs.

Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or "shock" drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection.IMPORTANCE The suggested "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats.

Histone H1 has seven variants in human somatic cells and contributes to chromatin compaction and transcriptional regulation. Knock-down (KD) of each H1 variant in breast cancer cells results in altered gene expression and proliferation differently in a variant specific manner with H1.2 and H1.4 KDs being most deleterious. Here we show combined depletion of H1.2 and H1.4 has a strong deleterious effect resulting in a strong interferon (IFN) response, as evidenced by an up-regulation of many IFN-stimulated genes (ISGs) not seen in individual nor in other combinations of H1 variant KDs. Although H1 participates to repress ISG promoters, IFN activation upon H1.2 and H1.4 KD is mainly generated through the activation of the IFN response by cytosolic nucleic acid receptors and IFN synthesis, and without changes in histone modifications at induced ISG promoters. H1.2 and H1.4 co-KD also promotes the appearance of accessibility sites genome wide and, particularly, at satellites and other repeats. The IFN response may be triggered by the expression of noncoding RNA generated from heterochromatic repeats or endogenous retroviruses upon H1 KD. In conclusion, redundant H1-mediated silencing of heterochromatin is important to maintain cell homeostasis and to avoid an unspecific IFN response.

p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.

SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

Specificities and genomic distribution of somatic mammalian histone H1 subtypes.

Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histone H1 family is evolutionarily diverse and many organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variants with a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, or whether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes.

Genome distribution of replication-independent histone H1 variants shows H1.0 associated with nucleolar domains and H1X associated with RNA polymerase II-enriched regions.

Unlike core histones, the linker histone H1 family is more evolutionarily diverse, and many organisms have multiple H1 variants or subtypes. In mammals, the H1 family includes seven somatic H1 variants; H1.1 to H1.5 are expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Using ChIP-sequencing data and cell fractionation, we have compared the genomic distribution of H1.0 and H1X in human breast cancer cells, in which we previously observed differential distribution of H1.2 compared with the other subtypes. We have found H1.0 to be enriched at nucleolus-associated DNA repeats and chromatin domains, whereas H1X is associated with coding regions, RNA polymerase II-enriched regions, and hypomethylated CpG islands. Further, H1X accumulates within constitutive or included exons and retained introns and toward the 3' end of expressed genes. Inducible H1X knockdown does not affect cell proliferation but dysregulates a subset of genes related to cell movement and transport. In H1X-depleted cells, the promoters of up-regulated genes are not occupied specifically by this variant, have a lower than average H1 content, and, unexpectedly, do not form an H1 valley upon induction. We conclude that H1 variants are not distributed evenly across the genome and may participate with some specificity in chromatin domain organization or gene regulation.

Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications.

In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues.