Changes In Chromatin Organization Research Articles

Cellular reprogramming for successful CNS axon regeneration is driven by a temporally changing cast of transcription factors

In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced “reprogramming” through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development.

Analyzing chromosome condensation in yeast by second-harmonic generation microscopy.

Condensation is a fundamental property of mitotic chromosomes in eukaryotic cells. However, analyzing chromosome condensation in yeast is a challenging task while existing methods have notable weaknesses. Second-harmonic generation (SHG) microscopy is a label-free, advanced imaging technique for measuring the surface curve of isotropic molecules such as chromatin in live cells. We applied this method to detect changes in chromatin organization throughout the cell cycle in live yeast cells. We showed that SHG microscopy can be used to identify changes in chromatin organization throughout the cell cycle and in response to inactivation of the SMC complexes, cohesin and condensin. Implementation of this method will improve our ability to analyze chromatin structure in protozoa and will enhance our understanding of chromatin organization in eukaryotic cells.

Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy.

Mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared with gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape segregation to the nuclear lamina and inactivation during cardiogenesis. These regions exhibit up-regulation of multiple noncardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. However, chromatin compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. Thus, global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.

Abstract 5177: Spatial co-fragmentation pattern of cell-free DNA recapitulates in vivo chromatin organization and identifies tissue-of-origin

Abstract Three-dimensional chromatin organization varies across different cell types and is essential for gene regulation. Functional genomic elements that reside kilobases to megabases away can be brought into spatial proximity by chromatin folding. In fixed cells, DNA fluorescence in situ hybridization and super-resolution microscopy can measure the distances between loci at ~10-100nm resolution, while chromosome conformation capture followed by next-generation sequencing (Hi-C) is able to profile genome-wide chromatin organization at kilobase-pair level resolution by measuring contact probabilities between pairs of loci. These methods provide a static snapshot of genome compaction and organization in different cellular states. However, assessing in vivo genome-wide chromatin organization changes non-invasively and longitudinally in patients is challenging due to the limitations of current technologies. Recently, circulating cell-free DNA (cfDNA) in blood has been shown as a promising biomarker to capture the genetic and local epigenetic changes within patients. Here, we inferred in vivo chromatin organization in blood cells from co-fragmentation patterns of cfDNA by using fragment lengths estimated from paired-end whole genome sequencing (WGS). We performed cfDNA WGS on 100 healthy, 34 colorectal cancer, 48 lung cancer, and 19 melanoma patients. The inferred chromatin organization is highly concordant with Hi-C performed on white blood cells and not explained by technical biases, sequence composition, or other epigenetic factors. Further, we developed methods to identify the tissue-of-origin of cfDNA based on its co-fragmentation pattern and Hi-C signal in reference cell types, which confirmed that most cfDNA in healthy individuals is derived from hematopoietic cells. In cancer patients, we observed an increased contribution to cfDNA from cancer cells that was quantitatively correlated with estimated tumor fraction in cfDNA and qualitatively matched tumor type. We also verified the results using publicly available cfDNA WGS data from different healthy and cancer patients. These results are consistent with previous studies that directly measured DNA methylation or that inferred nucleosome positions from WGS on cfDNA. However, our method has distinct advantages including using only low-coverage WGS, not requiring bisulfite treatment, and providing a more robust and quantitative estimation of cell type contributions. Collectively, our results demonstrate the potential of using cfDNA WGS to non-invasively assess the in vivo three-dimensional chromatin organization and determine tissue-of-origin in different physiological and pathological conditions, which may be useful for detecting, monitoring and treating different diseases. Citation Format: Yaping Liu, Tzu-Yu Liu, David Weinberg, Chris J. De La Torre, Catherine L. Tan, Anthony D. Schmitt, Siddarth Selvaraj, Vy Tran, Louise C. Laurent, François-Clément Bidard, Imran S. Haque. Spatial co-fragmentation pattern of cell-free DNA recapitulates in vivo chromatin organization and identifies tissue-of-origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5177.

Single Chromosome Aneuploidy Induces Genome-Wide Perturbation of Nuclear Organization and Gene Expression

Chromosomal aneuploidy is a defining feature of carcinomas and results in tumor-entity specific genomic imbalances. For instance, most sporadic colorectal carcinomas carry extra copies of chromosome 7, an aneuploidy that emerges already in premalignant adenomas, and is maintained throughout tumor progression and in derived cell lines. A comprehensive understanding on how chromosomal aneuploidy affects nuclear organization and gene expression, i.e., the nucleome, remains elusive. We now analyzed a cell line established from healthy colon mucosa with a normal karyotype (46,XY) and its isogenic derived cell line that acquired an extra copy of chromosome 7 as its sole anomaly (47,XY,+7). We studied structure/function relationships consequent to aneuploidization using genome-wide chromosome conformation capture (Hi-C), RNA sequencing and protein profiling. The gain of chromosome 7 resulted in an increase of transcript levels of resident genes as well as genome-wide gene and protein expression changes. The Hi-C analysis showed that the extra copy of chromosome 7 is reflected in more interchromosomal contacts between the triploid chromosomes. Chromatin organization changes are observed genome-wide, as determined by changes in A/B compartmentalization and topologically associating domain (TAD) boundaries. Most notably, chromosome 4 shows a profound loss of chromatin organization, and chromosome 14 contains a large A/B compartment switch region, concurrent with resident gene expression changes. No changes to the nuclear position of the additional chromosome 7 territory were observed when measuring distances of chromosome painting probes by interphase FISH. Genome and protein data showed enrichment in signaling pathways crucial for malignant transformation, such as the HGF/MET-axis. We conclude that a specific chromosomal aneuploidy has profound impact on nuclear structure and function, both locally and genome-wide. Our study provides a benchmark for the analysis of cancer nucleomes with complex karyotypes.

Determination of Chromatin Accessibility in Drosophila MidgutEnterocytes by in situ 5mC Labeling.

Regulation of gene expression involves dynamic changes in chromatin organization, where in many cases open chromatin structure correlates with gene activation. Several methods enable monitoring changes in chromatin accessibility, including ATAC-seq, FAIRE-seq, MNase-seq and DNAse-seq methods, which involve Next-generation-sequencing (NGS). Focusing on the adult Drosophila differentiated gut enterocytes (ECs) we used a sequencing-free method that enables visualizing and semi-quantifying large-scale changes in chromatin structure using in vitro methylation assay with the bacterial CpG Methyltransferase, M. Sssl, that determine chromatin accessibility. In brief, as CpG methylation is minimal in differentiated somatic Drosophila cells, we used the bacterial M. SssI enzyme to methylate CpG dinucleotides in situ depending on their chromatin accessibility. The methylated dinucleotides are detected using 5mCytosine monoclonal antibody and nuclei are visualized microscopically. Thus, the 5mC method enables to monitor large-scale chromatin changes in heterogenic cellular tissue focusing on the cell type of interest and without the need for cell purification or NGS.

Improved drug carriage and protective potential against Cisplatin-induced toxicity using Boldine-loaded PLGA nanoparticles

BackgroundCisplatin is a widely-used potent anti-cancer drug having severe side-effects precluding its sustained use. ObjectivesPoly (lactide-co-glycolide) (PLGA)-nanoparticles loaded Boldine, an antioxidant ingredient of ethanolic extract of Boldo plant (Peumus boldus) was tested in cancer mice model, Mus musculus to examine if it could reduce unwanted Cisplatin-induced toxicity in normal tissue. Material and methodsNano-encapsulation of Boldine was done by following the standardized solvent displacement method. Physico-chemical characterization of PLGA-encapsulated nano-Boldine (NBol) was accomplished through analyses of various spectroscopic techniques. Status of major antioxidant enzymes, functional markers, and lipid peroxidation (LPO) was also determined in certain tissue and serum samples. Percentage of cells undergoing cytotoxic death, Reactive oxygen species (ROS) accumulation and mitochondrial functioning were analyzed in both normal and cancer mice. Nanoscale changes in chromatin organization were assessed by Transmission electron microscopy (TEM). mRNA and protein expressions of Top II, Bax, Bcl-2, Cyt c, caspase 3 were studied by RT-PCR, immunoblot and immunofluorescence. ResultsNBol had faster mobility, site-specific action and ability of sustained particle release. NBol readily entered cells, prevented Cisplatin to intercalate with dsDNA resulting in reduction of chromatin condensation, with corresponding changes in ROS levels, mitochondrial functioning and antioxidant enzyme activities, leading to reduction in Deoxyribose nucleic acid (DNA) damage and cytotoxic cell death. Expression pattern of apoptotic genes like Top II, p53, Bax, Bcl-2, cytochrome c and caspase-3 suggested greater cytoprotective potentials of NBol in normal tissues. ConclusionsCompared to Boldine (Bol), NBol had better ability of drug carriage and protective potentials (29.00% approximately) against Cisplatin-induced toxicity. Combinational therapeutic use of PLGA-NBol can reduce unwanted Cisplatin-induced cellular toxicity facilitating use of Cisplatin.

Abstract 2998: Dynamic 3d chromosomal landscapes in acute leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of pediatric and 25% of adult ALL cases. Despite improved prognosis driven by the significant advances in the molecular understanding of T-ALL, the outcome of T-ALL patients with primary resistant and relapsed leukemia remains poor. Recent evidences have highlighted a role for changes in 3D chromatin architecture in cancer progression. Herein, for the first time we explored genomewide changes in chromatin organization in primary T-ALL samples relative to T cells from healthy donors. We show how the organization of topologically associated domains (TADs) influences key oncogenic and tumor-suppressive loci by modifying the promoter-enhancer landscape, deregulating the transcriptional program and ultimately causing disease progression. To comprehensively characterize the chromatin landscape of clinically relevant loci in T-ALL, we sought to identify changes in intra-TAD activity and TAD disruptions on a genome-wide scale. We developed and implemented new computational approaches for Hi-C data analysis with a focus on Hi-C data integration with ChIP-Seq and RNA-Seq. We found significant correlations of TAD activity with CTCF occupancy, super-enhancer activity and gene expression of leukemia relevant loci. Furthermore, among the dozens of TAD disruptions we identified, we found a novel TAD “fusion” event around the MYC locus, which allows for chromatin interactions between a recently reported Notch-dependent MYC super-enhancer and promoter to drive overexpression of MYC. This particular TAD fusion is accompanied by a complete CTCF loss in the TAD boundary and increased MYC expression in T-ALL samples. Overall, this study sheds new light into how changes in chromatin architecture influence disease progression by restructuring oncogene and tumor suppressor landscapes. Citation Format: Palaniraja Thandapani, Andreas Kloetgen, Charalampos Lazaris, Xufeng Chen, Panagiotis Ntziachristos, Aristotelis Tsirigos, Iannis Aifantis. Dynamic 3d chromosomal landscapes in acute leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2998.

HIF-dependent and reversible nucleosome disassembly in hypoxia-inducible gene promoters

Hypoxia causes dramatic changes in gene expression profiles, and the mechanism of hypoxia-inducible transcription has been analyzed for use as a model system of stress-inducible gene regulation. In this study, changes in chromatin organization in promoters of hypoxia-inducible genes were investigated during hypoxia-reoxygenation conditions. Most of the hypoxia-inducible gene promoters were hypersensitive to DNase I under both normal and hypoxic conditions, and our data indicate an immediate recruitment of transcription factors under hypoxic conditions. In some of the hypoxia-inducible promoters, nucleosome-free DNA regions (NFRs) were established in parallel with hypoxia-induced transcription. We also show that the hypoxia-inducible formation of NFRs requires that hypoxia-inducible transcription factors (HIFs) bind to the promoters together with the transcriptional coactivator CBP. Within 1 h after the hypoxia exposure was ended (reoxygenation), HIF complexes were dissociated from the promoter regions. Within 24 h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible NFRs. Nucleosome reassembly required the function of the transcriptional coregulator SIN3A. Thus, reversible changes in nucleosome organization mediated by transcription factors are notable features of stress-inducible gene regulation.

Neural stem cells from a mouse model of Rett syndrome are prone to senescence, show reduced capacity to cope with genotoxic stress, and are impaired in the differentiation process

Several aspects of stem cell life are governed by epigenetic variations, such as DNA methylation, histone modifications, and chromatin remodeling. Epigenetic events are also connected with the impairment of stem cell functions. For example, during senescence, there are significant changes in chromatin organization that alter transcription. The MECP2 protein can bind methylated cytosines and contribute to regulating gene expression at one of the highest hierarchical levels. Researchers are particularly interested in this protein, as up to 90% of Rett syndrome patients have an MECP2 gene mutation. Nevertheless, the role of MECP2 in this disease remains poorly understood. We used a mouse model of Rett syndrome to evaluate whether residual MECP2 activity in neural stem cells (NSCs) induced the senescence phenomena that could affect stem cell function. Our study clearly demonstrated that the reduced expression of MECP2 is connected with an increase in senescence, an impairment in proliferation capacity, and an accumulation of unrepaired DNA foci. Mecp2+/− NSCs did not cope with genotoxic stress in the same way as the control cells did. Indeed, after treatment with different DNA-damaging agents, the NSCs from mice with mutated Mecp2 accumulated more DNA damage foci (γ-H2AX+) and were more prone to cell death than the controls. Senescence in Mecp2+/− NSCs decreased the number of stem cells and progenitors and gave rise to a high percentage of cells that expressed neither stem/progenitor nor differentiation markers. These cells could be senescent and dysfunctional.

Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4+CD8+ Tcells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of Tcell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.

Epigenetic control of CD8+ T cell differentiation.

Upon stimulation, small numbers of naive CD8+ T cells proliferate and differentiate into a variety of memory and effector cell types. CD8+ T cells can persist for years and kill tumour cells and virally infected cells. The functional and phenotypic changes that occur during CD8+ T cell differentiation are well characterized, but the epigenetic states that underlie these changes are incompletely understood. Here, we review the epigenetic processes that direct CD8+ T cell differentiation and function. We focus on epigenetic modification of DNA and associated histones at genes and their regulatory elements. We also describe structural changes in chromatin organization that affect gene expression. Finally, we examine the translational potential of epigenetic interventions to improve CD8+ T cell function in individuals with chronic infections and cancer.

The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress

Age-associated changes in chromatin structure have a major impact on organismal longevity. Despite being a central part of the ageing process, the organismal responses to the changes in chromatin organization remain unclear. Here we show that moderate disturbance of histone balance during C. elegans development alters histone levels and triggers a stress response associated with increased expression of cytosolic small heat-shock proteins. This stress response is dependent on the transcription factor, HSF-1, and the chromatin remodeling factor, ISW-1. In addition, we show that mitochondrial stress during developmental stages also modulates histone levels, thereby activating a cytosolic stress response similar to that caused by changes in histone balance. These data indicate that histone and mitochondrial perturbations are both monitored through chromatin remodeling and involve the activation of a cytosolic response that affects organismal longevity. HSF-1 and ISW-1 hence emerge as a central mediator of this multi-compartment proteostatic response regulating longevity.

Chromatin organization changes during the establishment and maintenance of the postmitotic state

BackgroundGenome organization changes during development as cells differentiate. Chromatin motion becomes increasingly constrained and heterochromatin clusters as cells become restricted in their developmental potential. These changes coincide with slowing of the cell cycle, which can also influence chromatin organization and dynamics. Terminal differentiation is often coupled with permanent exit from the cell cycle, and existing data suggest a close relationship between a repressive chromatin structure and silencing of the cell cycle in postmitotic cells. Heterochromatin clustering could also contribute to stable gene repression to maintain terminal differentiation or cell cycle exit, but whether clustering is initiated by differentiation, cell cycle changes, or both is unclear. Here we examine the relationship between chromatin organization, terminal differentiation and cell cycle exit.ResultsWe focused our studies on the Drosophila wing, where epithelial cells transition from active proliferation to a postmitotic state in a temporally controlled manner. We find there are two stages of G0 in this tissue, a flexible G0 period where cells can be induced to reenter the cell cycle under specific genetic manipulations and a state we call “robust,” where cells become strongly refractory to cell cycle reentry. Compromising the flexible G0 by driving ectopic expression of cell cycle activators causes a global disruption of the clustering of heterochromatin-associated histone modifications such as H3K27 trimethylation and H3K9 trimethylation, as well as their associated repressors, Polycomb and heterochromatin protein 1 (HP1). However, this disruption is reversible. When cells enter a robust G0 state, even in the presence of ectopic cell cycle activity, clustering of heterochromatin-associated modifications is restored. If cell cycle exit is bypassed, cells in the wing continue to terminally differentiate, but heterochromatin clustering is severely disrupted. Heterochromatin-dependent gene silencing does not appear to be required for cell cycle exit, as compromising the H3K27 methyltransferase Enhancer of zeste, and/or HP1 cannot prevent the robust cell cycle exit, even in the face of normally oncogenic cell cycle activities.ConclusionsHeterochromatin clustering during terminal differentiation is a consequence of cell cycle exit, rather than differentiation. Compromising heterochromatin-dependent gene silencing does not disrupt cell cycle exit.

Abstract 2433: Altering nuclear size impacts cancer cell characteristics in melanoma cell lines

Abstract Nuclear size is altered in cancer cells, a change used by pathologists to distinguish cancer from normal cells. Previous studies in Xenopus identified two nuclear transport proteins, importin α and NTF2, as regulators of nuclear size. In general, importin α and NTF2 levels positively and negatively affect nuclear size, respectively. Increased importin α expression is used as a biomarker in non-small cell lung carcinoma and breast cancer and correlates with increased nuclear size. We identified decreased NTF2 protein expression as a potential biomarker in melanoma, consistent with NTF2 levels negatively regulating nuclear size. Following up on this result, our current studies focus on manipulating nuclear size in melanoma cells to assess the impact on cancer cell characteristics, including proliferation rate, migration potential, and apoptosis. First, we measured nuclear size and NTF2 expression levels in different stage melanoma cell lines. Compared to a normal melanocyte cell line, we consistently observed larger nuclei and lower NTF2 protein levels in all melanoma cell lines examined. Next, we performed transient transfections in HeLa, MRC5, and melanoma cell lines, demonstrating that ectopic NTF2 expression leads to reduced nuclear size. The most dramatic effects were observed in primary melanoma cell line WM3211 where higher NTF2 expression caused a 40% reduction in nuclear cross-sectional area. To obtain more precise control over NTF2 expression levels, we generated a stably transfected metastatic melanoma cell line (WM983B) in which NTF2 expression can be titrated by varying the concentration of doxycycline in the growth media. Higher doxycycline levels lead to increased NTF2 expression and smaller nuclei. In particular, 20 ng/ml doxycycline induced a 10% reduction in nuclear cross-sectional area. In a wound healing assay, doxycycline-treated cells exhibited a 40% reduction in cell motility compared to non-treated cells or the parent cell line treated with doxycycline. Furthermore, the doxycycline-treated cells exhibited an increased rate of apoptosis. We propose that reduced nuclear size in the doxycycline-treated cells causes changes in chromatin organization and gene expression, thus giving rise to observed effects on cell migration and apoptosis. To test this idea, we are currently performing a global transcriptomics analysis of our cell lines to identify genes whose expression is altered by nuclear size. Using this information, we will use DNA FISH to map the intranuclear position of differentially-expressed genes as a function of nuclear size. Also in vivo experiments are ongoing in which we have subcutaneously injected NTF2-inducible melanoma cells into NSG mice in order to examine tumor formation and metastatic capacity of cells with differently-sized nuclei. Regarding that in most cancer cells nuclear size is enlarged, understanding the causes and effects of these changes can help us to better understand cancer biology. Citation Format: Lidija D. Vukovic, Bradley A. Stohr, Dan L. Levy. Altering nuclear size impacts cancer cell characteristics in melanoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2433. doi:10.1158/1538-7445.AM2017-2433

Nucleosome repositioning during differentiation of a human myeloid leukemia cell line

ABSTRACTCell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.

Reciprocal regulation of chromatin state and architecture by HOTAIRM1 contributes to temporal collinear HOXA gene activation.

Thousands of long non-coding RNAs (lncRNAs) have been identified in mammals, many of which represent important regulators of gene expression. However, the mechanisms used by lncRNAs to control transcription remain largely uncharacterized. Here, we report on HOTAIRM1, a promising lncRNA biomarker in leukemia and solid tumors. We find that HOTAIRM1 contributes to three-dimensional chromatin organization changes required for the temporal collinear activation of HOXA genes. We show that distinct HOTAIRM1 variants preferentially associate with either UTX/MLL or PRC2 complexes to modulate the levels of activating and silencing marks at the bivalent domain. HOTAIRM1 contributes to physical dissociation of chromatin loops at the cluster proximal end, which delays recruitment of the histone demethylase UTX and transcription of central HOXA genes. Interestingly, we find overall proximal HOXA gene activation without chromatin conformation changes by HOTAIRM1 in a different cell type. Our results reveal a previously unappreciated relationship between chromatin structure, architecture and lncRNA function.

Epigenetic marks in the Hyacinthus orientalis L. mature pollen grain and during in vitro pollen tube growth.

During the sexual reproduction of flowering plants, epigenetic control of gene expression and genome integrity by DNA methylation and histone modifications plays an important role in male gametogenesis. In this study, we compared the chromatin modification patterns of the generative, sperm cells and vegetative nuclei during Hyacinthus orientalis male gametophyte development. Changes in the spatial and temporal distribution of 5-methylcytosine, acetylated histone H4 and histone deacetylase indicated potential differences in the specific epigenetic state of all analysed cells, in both the mature cellular pollen grains and the in vitro growing pollen tubes. Interestingly, we observed unique localization of chromatin modifications in the area of the generative and the vegetative nuclei located near each other in the male germ unit, indicating the precise mechanisms of gene expression regulation in this region. We discuss the differences in the patterns of the epigenetic marks along with our previous reports of nuclear metabolism and changes in chromatin organization and activity in hyacinth male gametophyte cells. We also propose that this epigenetic status of the analysed nuclei is related to the different acquired fates and biological functions of these cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00497-016-0289-3) contains supplementary material, which is available to authorized users.

HMGN proteins modulate chromatin regulatory sites and gene expression during activation of naïve B cells.

The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn−/− mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes.

The histone methyltransferase Setd7 promotes pancreatic progenitor identity.

Cell fate specification depends on transcriptional activation driven by lineage-specific transcription factors as well as changes in chromatin organization. To date, the interplay between transcription factors and chromatin modifiers during development is not well understood. We focus here on the initiation of the pancreatic program from multipotent endodermal progenitors. Transcription factors that play key roles in regulating pancreatic progenitor state have been identified, but the chromatin regulators that help to establish and maintain pancreatic fate are less well known. Using a comparative approach, we identify a crucial role for the histone methyltransferase Setd7 in establishing pancreatic cell identity. We show that Setd7 is expressed in the prospective pancreatic endoderm of Xenopus and mouse embryos prior to Pdx1 induction. Importantly, we demonstrate that setd7 is sufficient and required for pancreatic cell fate specification in Xenopus Functional and biochemical approaches in Xenopus and mouse endoderm support that Setd7 modulates methylation marks at pancreatic regulatory regions, possibly through interaction with the transcription factor Foxa2. Together, these results demonstrate that Setd7 acts as a central component of the transcription complex initiating the pancreatic program.