In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

LATS1 controls CTCF chromatin occupancy and hormonal response of 3D-grown breast cancer cells.

The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.

The post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes. Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.

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.

ATP, Mg2+, Nuclear Phase Separation, and Genome Accessibility.

Misregulation of the processes controlling eukaryotic gene expression can result in disease. Gene expression is influenced by the surrounding chromatin; hence the nuclear environment is also of vital importance. Recently, understanding of chromatin hierarchical folding has increased together with the discovery of membrane-less organelles which are distinct, dynamic liquid droplets that merge and expand within the nucleus. These 'sieve'-like regions may compartmentalize and separate functionally distinct regions of chromatin. This article aims to discuss recent studies on nuclear phase within the context of poly(ADP-ribose), ATP, and Mg2+ levels, and we propose a combinatorial complex role for these molecules in phase separation and genome regulation. We also discuss the implications of this process for gene regulation and discuss possible strategies to test this.

C/EBPα mediates the growth inhibitory effect of progestins on breast cancer cells.

Steroid hormones are key gene regulators in breast cancer cells. While estrogens stimulate cell proliferation, progestins activate a single cell cycle followed by proliferation arrest. Here, we use biochemical and genome-wide approaches to show that progestins achieve this effect via a functional crosstalk with C/EBPα. Using ChIP-seq, we identify around 1,000 sites where C/EBPα binding precedes and helps binding of progesterone receptor (PR) in response to hormone. These regions exhibit epigenetic marks of active enhancers, and C/EBPα maintains an open chromatin conformation that facilitates loading of ligand-activated PR. Prior to hormone exposure, C/EBPα favors promoter-enhancer contacts that assure hormonal regulation of key genes involved in cell proliferation by facilitating binding of RAD21, YY1, and the Mediator complex. Knockdown of C/EBPα disrupts enhancer-promoter contacts and decreases the presence of these architectural proteins, highlighting its key role in 3D chromatin looping. Thus, C/EBPα fulfills a previously unknown function as a potential growth modulator in hormone-dependent breast cancer.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations.

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.

Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock.

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.

Hormone-control regions mediate steroid receptor-dependent genome organization.

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.

Distinct structural transitions of chromatin topological domains correlate with coordinated hormone-induced gene regulation.

The human genome is segmented into topologically associating domains (TADs), but the role of this conserved organization during transient changes in gene expression is not known. Here we describe the distribution of progestin-induced chromatin modifications and changes in transcriptional activity over TADs in T47D breast cancer cells. Using ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing), Hi-C (chromosome capture followed by high-throughput sequencing), and three-dimensional (3D) modeling techniques, we found that the borders of the ∼ 2000 TADs in these cells are largely maintained after hormone treatment and that up to 20% of the TADs could be considered as discrete regulatory units where the majority of the genes are either transcriptionally activated or repressed in a coordinated fashion. The epigenetic signatures of the TADs are homogeneously modified by hormones in correlation with the transcriptional changes. Hormone-induced changes in gene activity and chromatin remodeling are accompanied by differential structural changes for activated and repressed TADs, as reflected by specific and opposite changes in the strength of intra-TAD interactions within responsive TADs. Indeed, 3D modeling of the Hi-C data suggested that the structure of TADs was modified upon treatment. The differential responses of TADs to progestins and estrogens suggest that TADs could function as "regulons" to enable spatially proximal genes to be coordinately transcribed in response to hormones.

Nucleosome-driven transcription factor binding and gene regulation.

Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions identified as DNaseI-hypersensitive sites (DHS). Here we show by ChIP, MNase, and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins, we identified 25,000 PR binding sites (PRbs). The majority of these sites encompassed several copies of the hexanucleotide TGTYCY, which is highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes, mainly in enhancers. Most of these sites overlap with DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation.

Unliganded progesterone receptor-mediated targeting of an RNA-containing repressive complex silences a subset of hormone-inducible genes.

A close chromatin conformation precludes gene expression in eukaryotic cells. Genes activated by external cues have to overcome this repressive state by locally changing chromatin structure to a more open state. Although much is known about hormonal gene activation, how basal repression of regulated genes is targeted to the correct sites throughout the genome is not well understood. Here we report that in breast cancer cells, the unliganded progesterone receptor (PR) binds genomic sites and targets a repressive complex containing HP1γ (heterochromatin protein 1γ), LSD1 (lysine-specific demethylase 1), HDAC1/2, CoREST (corepressor for REST [RE1 {neuronal repressor element 1} silencing transcription factor]), KDM5B, and the RNA SRA (steroid receptor RNA activator) to 20% of hormone-inducible genes, keeping these genes silenced prior to hormone treatment. The complex is anchored via binding of HP1γ to H3K9me3 (histone H3 tails trimethylated on Lys 9). SRA interacts with PR, HP1γ, and LSD1, and its depletion compromises the loading of the repressive complex to target chromatin-promoting aberrant gene derepression. Upon hormonal treatment, the HP1γ-LSD1 complex is displaced from these constitutively poorly expressed genes as a result of rapid phosphorylation of histone H3 at Ser 10 mediated by MSK1, which is recruited to the target sites by the activated PR. Displacement of the repressive complex enables the loading of coactivators needed for chromatin remodeling and activation of this set of genes, including genes involved in apoptosis and cell proliferation. These results highlight the importance of the unliganded PR in hormonal regulation of breast cancer cells.

OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes.

The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb.

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells.

Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer.

Signaling by Steroid Hormones in the 3D Nuclear Space.

Initial studies showed that ligand-activated hormone receptors act by binding to the proximal promoters of individual target genes. Genome-wide studies have now revealed that regulation of transcription by steroid hormones mainly depends on binding of the receptors to distal regulatory elements. Those distal elements, either enhancers or silencers, act on the regulation of target genes by chromatin looping to the gene promoters. In the nucleus, this level of chromatin folding is integrated within dynamic higher orders of genome structures, which are organized in a non-random fashion. Terminally differentiated cells exhibit a tissue-specific three-dimensional (3D) organization of the genome that favors or restrains the activity of transcription factors and modulates the function of steroid hormone receptors, which are transiently activated upon hormone exposure. Conversely, integration of the hormones signal may require modifications of the 3D organization to allow appropriate transcriptional outcomes. In this review, we summarize the main levels of organization of the genome, review how they can modulate the response to steroids in a cell specific manner and discuss the role of receptors in shaping and rewiring the structure in response to hormone. Taking into account the dynamics of 3D genome organization will contribute to a better understanding of the pleiotropic effects of steroid hormones in normal and cancer cells.

Two chromatin remodeling activities cooperate during activation of hormone responsive promoters.

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Nuclear factor 1 synergizes with progesterone receptor on the mouse mammary tumor virus promoter wrapped around a histone H3/H4 tetramer by facilitating access to the central hormone-responsive elements.

Steroid hormones induce transcription of their responsive genes by complex mechanisms including synergism between the hormone receptors and other transcription factors. On the mouse mammary tumor virus (MMTV) promoter progesterone induction is mediated by the reciprocal synergism between progesterone receptor (PR) and the ubiquitous transcription factor nuclear factor 1 (NF1). PR binding mediates ATP-dependent displacement of histone H2A and H2B, enabling NF1 access to its target site. In minichromosomes assembled in vitro NF1 binding facilitates access of PR to the hormone-responsive elements (HREs) by precluding reforming of the histone octamer, but the function of NF1 in living cells remains unclear. Here we show that depleting NF1 by small interfering RNAs or mutating the NF1-binding site significantly compromises transcription of the MMTV promoter. The central HREs 2 and 3 are not needed for ATP-dependent H2A/H2B displacement or NF1 binding but are critical for full PR binding and MMTV transactivation. We found that NF1 binding to the MMTV promoter on a H3/H4 histone tetramer particle exposes the central HREs and facilitates their binding by PR, suggesting a possible mechanism for the reciprocal synergism between PR and NF1.

MyoD induces ARTD1 and nucleoplasmic poly-ADP-ribosylation during fibroblast to myoblast transdifferentiation.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.