Heterochromatic Regions Research Articles

Lamins and chromatin join forces

The intricate interplay between lamins and chromatin underpins the structural integrity and functional organization of the eukaryotic nucleus. Lamins, type V intermediate filament proteins, form a robust meshwork beneath the inner nuclear membrane that is crucial for sustaining nuclear architecture through interactions with lamin-associated domains (LADs). LADs are predominantly heterochromatic regions in which compacted chromatin is enriched at the nuclear periphery, interacting with lamins and lamin-associated proteins. Disruptions of these interactions are implicated in a spectrum of diseases, including laminopathies, cancer, and age-related pathologies, highlighting the importance of lamin-LAD interactions. Thus, a detailed understanding of lamin-chromatin interactions may provide new insights into chromatin organization and shed light on the mechanism behind certain disease states. Here, we discuss the current state of knowledge of lamin-chromatin interactions from a biochemical and structural point of view.

Species-specific satellite DNA composition dictates de novo formation of PRC1-mediated pericentric heterochromatin.

Pericentromeres are heterochromatic regions adjacent to centromeres that ensure accurate chromosome segregation. Despite their conserved function, they are composed of rapidly evolving A/T-rich satellite DNA. This paradoxical observation is partially resolved by epigenetic mechanisms that maintain heterochromatin, independent of specific DNA sequences. However, it is unclear how satellite DNA sequence variation impacts de novo formation of pericentric heterochromatin, which is initially absent from paternal chromosomes in the zygote. Here we show that satellite variation has functional consequences for zygotic heterochromatin formation, recruitment of the Chromosome Passenger Complex (CPC), and interactions with spindle microtubules. In M. musculus zygotes, Polycomb Repressive Complex 1 (PRC1) is recruited to pericentric satellites by its AT-hook domain, which binds runs of A/T nucleotides, to generate H2AK119ub1 (H2Aub) heterochromatin. By fertilizing M. musculus eggs with sperm from other mouse species, we show that species-specific satellite sequences differ in their ability to recruit PRC1 and form H2Aub. This satellite-DNA mediated increase in PRC1 heterochromatin leads to reduced CPC recruitment and increased microtubule forces on kinetochores. Our results provide a direct link between satellite DNA composition and pericentromere function in the zygote, when epigenetic pathways maintaining pericentromere heterochromatin are absent.

LAP2alpha facilitates myogenic gene expression by preventing nucleoplasmic lamin A/C from spreading to active chromatin regions.

A-type lamins form a filamentous meshwork beneath the nuclear membrane that anchors large heterochromatic genomic regions at the nuclear periphery. A-type lamins also exist as a dynamic, non-filamentous pool in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Both proteins associate with largely overlapping euchromatic genomic regions in the nucleoplasm, but the functional significance of this interaction is poorly understood. Here, we report that LAP2α relocates towards regions containing myogenic genes in the early stages of muscle differentiation, possibly facilitating efficient gene regulation, while lamins A and C mostly associate with genomic regions away from these genes. Strikingly, upon depletion of LAP2α, A-type lamins spread across active chromatin and accumulate at regions of active H3K27ac and H3K4me3 histone marks in the vicinity of myogenic genes whose expression is impaired in the absence of LAP2α. Reorganization of A-type lamins on chromatin is accompanied by depletion of the active chromatin mark H3K27ac and a significantly impaired myogenic differentiation. Thus, the interplay of LAP2α and A-type lamins is crucial for proper positioning of intranuclear lamin A/C on chromatin to allow efficient myogenic differentiation.

Klinefelter syndrome in a patient with double Y-autosomal translocation

Klinefelter syndrome is one of the most common chromosomal abnormalities and the most common genetic cause of male infertility. About 85 % of patients have 47,XXY karyotype, other patients have other non-mosaic and mosaic Klinefelter syndrome variants. We report a unique clinical case — Klinefelter syndrome patient with double Y autosomal translocation. The proband is a 15-year-old male patient (height 180 cm, weight 50 kg, normal IQ) who was admitted for cytogenetic examination and genetic counseling due to delayed puberty. He was diagnosed with testicular hypoplasia, hypergonadotropic hypogonadism, pituitary microadenoma and left-sided varicocele. The proband was born in a nonconsanguineous marriage after in vitro fertilization due to paternal male factor infertility. Cytogenetic analysis was performed on cultured peripheral blood lymphocytes using standard chromosome analysis with GTG staining and FISH analysis. Molecular analysis of the Y chromosome was performed by multiplex PCR. Complex cytogenetic examination revealed a 46,XX,der(Y) t(Y;15)(q12;q11.1),der(13)t(Y;13)(q12;p11.2),-15 karyotype in the proband. Molecular analysis showed that the proband is SRY positive; no microdeletion of the Y chromosome was found. The detected double Y autosomal translocation is a chromosomal abnormality independent of KS. The father of the proband is an oligozoospermic man with robertsonian translocation 13;15 — 45,XY,der(13;15)(q10;q10), the mother has a normal karyotype 46,XX. Apparently, the der(13) and der(Y) chromosomes result from abnormal meiotic recombination in paternal meiosis between the heterochromatic region Yq12 and the centromeric/pericentromeric heterochromatin of chromosomes 13 and 15 involved in the paternal robertsonian translocation, and Klinefelter syndrome is due to the presence of two X chromosomes in the karyotype in the presence of a derivative Y chromosome. The detected double Y-autosomal translocation is a chromosomal abnormality unrelated to Klinefelter syndrome, arising on the background of the paternal robertsonian translocation.

Developmental analysis of Spalt function in the Drosophila prothoracic gland.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance

Heterochromatin enforces transcriptional gene silencing and can be epigenetically inherited, but the underlying mechanisms remain unclear. Here, we show that histone deacetylation, a conserved feature of heterochromatin domains, blocks SWI/SNF subfamily remodelers involved in chromatin unraveling, thereby stabilizing modified nucleosomes that preserve gene silencing. Histone hyperacetylation, resulting from either the loss of histone deacetylase (HDAC) activity or the direct targeting of a histone acetyltransferase to heterochromatin, permits remodeler access, leading to silencing defects. The requirement for HDAC in heterochromatin silencing can be bypassed by impeding SWI/SNF activity. Highlighting the crucial role of remodelers, merely targeting SWI/SNF to heterochromatin, even in cells with functional HDAC, increases nucleosome turnover, causing defective gene silencing and compromised epigenetic inheritance. This study elucidates a fundamental mechanism whereby histone hypoacetylation, maintained by high HDAC levels in heterochromatic regions, ensures stable gene silencing and epigenetic inheritance, providing insights into genome regulatory mechanisms relevant to human diseases.

ModDotPlot-rapid and interactive visualization of tandem repeats.

A common method for analyzing genomic repeats is to produce a sequence similarity matrix visualized via a dot plot. Innovative approaches such as StainedGlass have improved upon this classic visualization by rendering dot plots as a heatmap of sequence identity, enabling researchers to better visualize multi-megabase tandem repeat arrays within centromeres and other heterochromatic regions of the genome. However, computing the similarity estimates for heatmaps requires high computational overhead and can suffer from decreasing accuracy. In this work, we introduce ModDotPlot, an interactive and alignment-free dot plot viewer. By approximating average nucleotide identity via a k-mer-based containment index, ModDotPlot produces accurate plots orders of magnitude faster than StainedGlass. We accomplish this through the use of a hierarchical modimizer scheme that can visualize the full 128 Mb genome of Arabidopsis thaliana in under 5 min on a laptop. ModDotPlot is bundled with a graphical user interface supporting real-time interactive navigation of entire chromosomes. ModDotPlot is available at https://github.com/marbl/ModDotPlot.

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs)with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa.

Genome organization is essential for proper function, including gene expression. In metazoan genome organization, chromatin loops and Topologically Associated Domains (TADs) facilitate local gene clustering, while chromosomes form distinct nuclear territories characterized by compartmentalization of silent heterochromatin at the nuclear periphery and active euchromatin in the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation, where heterochromatic centromeres and telomeres independently cluster at the nuclear membrane, while interspersed heterochromatic loci in Neurospora aggregate across megabases of linear genomic distance for forming TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. Here, we examined the genome organization of a Neurospora strain harboring a ~47.4 kilobase facultative (temporarily silent) heterochromatic region deletion, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion had little effect on local chromatin structure, the constitutive heterochromatin deletion altered local TAD-like structures, gene expression, and the predicted 3D genome structure by qualitatively repositioning genes into the nucleus center. Our work elucidates the role of individual heterochromatic regions for genome organization and function.

Mechanism of heterochromatin remodeling revealed by the DDM1 bound nucleosome structures

The SWI/SNF2 chromatin remodeling factor decreased DNA methylation 1 (DDM1) is essential for the silencing of transposable elements (TEs) in both euchromatic and heterochromatic regions. Here, we determined the cryo-EM structures of DDM1-nucleosomeH2A and DDM1-nucleosomeH2A.W complexes at near-atomic resolution in the presence of the ATP analog ADP-BeFx. The structures show that nucleosomal DNA is unwrapped more on the surface of the histone octamer containing histone H2A than that containing histone H2A.W. DDM1 embraces one DNA gyre of the nucleosome and interacts with the N-terminal tails of histone H4. Although we did not observe DDM1-H2A.W interactions in our structures, the results of the pull-down experiments suggest a direct interaction between DDM1 and the core region of histone H2A.W. Our work provides mechanistic insights into the heterochromatin remodeling process driven by DDM1 in plants.

Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase

BackgroundEpigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1.PurposeHere, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system.ResultsMethylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification.ConclusionCollectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles.

Chromosome mapping of retrotransposon AviRTE in a neotropical bird species: Trogon surrucura (Trogoniformes; Trogonidae).

Avian genomes are characterized as being more compact than other amniotes, with less diversity and density of transposable elements (TEs). In addition, birds usually show bimodal karyotypes, exhibiting a great variation in diploid numbers. Some species present unusually large sex chromosomes, possibly due to the accumulation of repetitive sequences. Avian retrotransposon-like element (AviRTE) is a long interspersed nuclear element (LINE) recently discovered in the genomes of birds and nematodes, and it is still poorly characterized in terms of chromosomal mapping and phylogenetic relationships. In this study, we mapped AviRTE isolated from the Trogon surrucura genome into the T. surrucura (TSU) karyotype. Furthermore, we analyzed the phylogenetic relationships of this LINE in birds and other vertebrates. Our results showed that the distribution pattern of AviRTE is not restricted to heterochromatic regions, with accumulation on the W chromosome of TSU, yet another species with an atypical sex chromosome and TE hybridization. The phylogenetic analysis of AviRTE sequences in birds agreed with the proposed phylogeny of species in most clades, and allowed the detection of this sequence in other species, expanding the distribution of the element.

Epigenetic marks uniquely tune the material properties of HP1α condensates

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α condensates, whereas peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1α phase separation. Using fluorescence microscopy and rheological approaches, we further demonstrate that H3K9me3 histone peptides modulate the dynamics and viscoelastic network properties of HP1α condensates in a concentration-dependent manner. Additionally, in cells exposed to uniaxial strain, we find there to be a decreased ratio of nuclear H3K9me3 to HP1α. These data suggest that HP1α-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells and in response to mechanostimulation.

Non-canonical bases differentially represented in the sex chromosomes of the dioecious plant Silene latifolia.

The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin. This study utilizes Silene latifolia, a dioecious plant with heteromorphic sex chromosomes and a genome with a large proportion of TEs, which provides a favourable environment for the study of oxi-mCs in individual sexes. Notably, we detected surprisingly high levels of oxi-mCs in S. latifolia comparable with mammals. Nuclei showed enrichment in heterochromatic regions, except for 5hmC whose signal was homogeneously distributed. Intriguingly, the same X chromosome in females displayed overall enrichment of 5hmC and 5fC compared with its counterpart. This fact is shared with 5mC, resembling dosage compensation. Co-localization showed higher correlation between 5mC and 5fC than with 5hmC, indicating no potential relationship between 5hmC and 5fC. Additionally, the promoter of several sex-linked genes and sex-biased TEs clustered in a clear sex-dependent way. Together, these findings unveil a hypothetical role for oxi-mCs in S. latifolia sex chromosome development, warranting further exploration.

<i>Otu</i> and <i>Rif<sup>1</sup></i> double mutant enables analysis of satellite DNA in polytene chromosomes of ovarian germ cells in <i>Drosophila melanogaster</i>

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation were similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Two-way feedback between chromatin compaction and histone modification state explains Saccharomyces cerevisiae heterochromatin bistability

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae, that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins.

Interspecific cytogenomic comparison reveals a potential chromosomal centromeric marker in Proceratophrys frog species.

Among the repetitive elements, satellite DNA (SatDNA) emerges as extensive arrays of highly similar tandemly repeated units, spanning megabases in length. Given that the satDNA PboSat01-176, previously characterized in P. boiei, prompted our interest for having a high abundance in P. boiei and potential for centromeric satellite, here, we employed various approaches, including low coverage genome sequencing, followed by computational analysis and chromosomal localization techniques in four Proceratophrys species and, investigating the genomic presence and sharing, as well as its potential for chromosomal centromere marker in Proceratophrys frog species. Our findings demonstrate that PboSat01-176 exhibits high abundance across all four Proceratophrys species, displaying distinct characteristics that establish it as the predominant repetitive DNA element in these species. The satellite DNA is prominently clustered in the peri/centromeric region of the chromosomes, particularly in the heterochromatic regions. The widespread presence of PboSat01-176 in closely related Proceratophrys species reinforces the validity of the library hypothesis for repetitive sequences. Thus, this study highlighted the utility of the satDNA family PboSat01-176 as a reliable centromeric marker in Proceratophrys species, with potential to be applied in other species of anuran amphibians. The observed sharing and maintenance of this sequence within the genus suggest possibilities for future research, particularly through expanded sampling to elucidate parameters that underlie the library hypothesis and the evolutionary dynamics of satDNA sequences.

Abstract 3009: ATRX loss blocks the differentiation of mesenchymal stem cells and promotes undifferentiated sarcoma development

Abstract Soft tissue sarcomas (STSs) are a collection of rare tumor types originated from the mesenchymal tissues. Among the over 70 histological subtypes of STS, undifferentiated sarcoma (US), which lacks any discernible morphological features of differentiated tissues, accounts for over 20% of all STSs and is correlated with high tumor grade and poor prognosis. Recent genomic profiling in US has identified p53 and ATRX among the most frequently mutated or deleted genes. ATRX is a SWI/SNF2 type of chromatin remodeling factor which functions in a histone variant H3.3-specific chaperone complex to orchestrates replication-independent nucleosome assembly at repetitive heterochromatic DNA regions including telomeres. Loss-of-function mutations occur frequently in many tumor types and is associated with the development of alternative lengthening of telomere (ALT). However, how loss of ATRX function may contribute to tumorigenesis is largely unknown. Here, we demonstrated that ATRX is required for the terminal differentiation of mouse mesenchymal stem cells (MSCs) into multiple lineages, such as adipocytes and osteocytes. Targeted deletion of Atrx in mouse MSCs, but not terminally differentiated mesenchymal lineage cells, accelerated sarcoma initiation induced by loss of p53 and significantly shortened the overall survival. Atrx-deficient tumors express high level of MSC markers such as CD44 and CD29 and exhibit pathological features highly reminiscent of human high-grade US. Transcriptomic analysis indicates that Atrx deficiency is associated with enhanced proliferation. Together, our data indicates that ATRX deficiency contributes to undifferentiated sarcoma development by inhibiting mesenchymal stem cell differentiation. Citation Format: Yiming Cai, Jun Yao, Dean N. Pan, Yanqiang Jin, James M. You, Wantong Yao, Haoqiang Ying. ATRX loss blocks the differentiation of mesenchymal stem cells and promotes undifferentiated sarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3009.

Opposite and Differently Altered Postmortem Changes in H3 and H3K9me3 Patterns in the Rat Frontal Cortex and Hippocampus.

Temporal and spatial epigenetic modifications in the brain occur during ontogenetic development, pathophysiological disorders, and aging. When epigenetic marks, such as histone methylations, in brain autopsies or biopsy samples are studied, it is critical to understand their postmortem/surgical stability. For this study, the frontal cortex and hippocampus of adult rats were removed immediately (controls) or after a postmortem delay of 15, 30, 60, 90, 120, or 150 min. The patterns of unmodified H3 and its trimethylated form H3K9me3 were analyzed in frozen samples for Western blot analysis and in formalin-fixed tissues embedded in paraffin for confocal microscopy. We found that both the unmodified H3 and H3K9me3 showed time-dependent but opposite changes and were altered differently in the frontal cortex and hippocampus with respect to postmortem delay. In the frontal cortex, the H3K9me3 marks increased approximately 450% with a slow parallel 20% decrease in the unmodified H3 histones after 150 min. In the hippocampus, the change was opposite, since H3K9me3 marks decreased steadily by approximately 65% after 150 min with a concomitant rapid increase of 20-25% in H3 histones at the same time. Confocal microscopy located H3K9me3 marks in the heterochromatic regions of the nuclei of all major cell types in the control brains: oligodendrocytes, astrocytes, neurons, and microglia. Therefore, epigenetic marks could be affected differently by postmortem delay in different parts of the brain.

A function of spalt major as a sequence-specific DNA binding transcription factor mediates repression of knirps in the Drosophila wing imaginal disc

The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are unknow. Here, we analyze a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and we identified a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.