70 research outputs found
Genomic Prevalence of Heterochromatic H3K9me2 and Transcription Do Not Discriminate Pluripotent from Terminally Differentiated Cells
Cellular differentiation entails reprogramming of the transcriptome from a
pluripotent to a unipotent fate. This process was suggested to coincide with a
global increase of repressive heterochromatin, which results in a reduction of
transcriptional plasticity and potential. Here we report the dynamics of the
transcriptome and an abundant heterochromatic histone modification,
dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal
differentiation of embryonic stem cells. In contrast to the prevailing model, we
find H3K9me2 to occupy over 50% of chromosomal regions already in stem
cells. Marked are most genomic regions that are devoid of transcription and a
subgroup of histone modifications. Importantly, no global increase occurs during
differentiation, but discrete local changes of H3K9me2 particularly at genic
regions can be detected. Mirroring the cell fate change, many genes show altered
expression upon differentiation. Quantitative sequencing of transcripts
demonstrates however that the total number of active genes is equal between stem
cells and several tested differentiated cell types. Together, these findings
reveal high prevalence of a heterochromatic mark in stem cells and challenge the
model of low abundance of epigenetic repression and resulting global basal level
transcription in stem cells. This suggests that cellular differentiation entails
local rather than global changes in epigenetic repression and transcriptional
activity
The Mutant Form of Lamin A that Causes Hutchinson-Gilford Progeria Is a Biomarker of Cellular Aging in Human Skin
Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals
Role of the Epigenetic Regulator HP1γ in the Control of Embryonic Stem Cell Properties
The unique properties of embryonic stem cells (ESC) rely on long-lasting self-renewal and their ability to switch in all adult cell type programs. Recent advances have shown that regulations at the chromatin level sustain both ESC properties along with transcription factors. We have focused our interest on the epigenetic modulator HP1γ (Heterochromatin Protein 1, isoform γ) that binds histones H3 methylated at lysine 9 (meH3K9) and is highly plastic in its distribution and association with the transcriptional regulation of specific genes during cell fate transitions. These characteristics of HP1γ make it a good candidate to sustain the ESC flexibility required for rapid program changes during differentiation. Using RNA interference, we describe the functional role of HP1γ in mouse ESC. The analysis of HP1γ deprived cells in proliferative and in various differentiating conditions was performed combining functional assays with molecular approaches (RT-qPCR, microarray). We show that HP1γ deprivation slows down the cell cycle of ESC and decreases their resistance to differentiating conditions, rendering the cells poised to differentiate. In addition, HP1γ depletion hampers the differentiation to the endoderm as compared with the differentiation to the neurectoderm or the mesoderm. Altogether, our results reveal the role of HP1γ in ESC self-renewal and in the balance between the pluripotent and the differentiation programs
Epigenetic control of nuclear architecture
The cell nucleus is a highly structured compartment where nuclear components are thought to localize in non-random positions. Correct positioning of large chromatin domains may have a direct impact on the localization of other nuclear components, and can therefore influence the global functionality of the nuclear compartment. DNA methylation of cytosine residues in CpG dinucleotides is a prominent epigenetic modification of the chromatin fiber. DNA methylation, in conjunction with the biochemical modification pattern of histone tails, is known to lock chromatin in a close and transcriptionally inactive conformation. The relationship between DNA methylation and large-scale organization of nuclear architecture, however, is poorly understood. Here we briefly summarize present concepts of nuclear architecture and current data supporting a link between DNA methylation and the maintenance of large-scale nuclear organization
Validation of Skeletal Muscle cis-Regulatory Module Predictions Reveals Nucleotide Composition Bias in Functional Enhancers
We performed a genome-wide scan for muscle-specific cis-regulatory modules (CRMs) using three computational prediction programs. Based on the predictions, 339 candidate CRMs were tested in cell culture with NIH3T3 fibroblasts and C2C12 myoblasts for capacity to direct selective reporter gene expression to differentiated C2C12 myotubes. A subset of 19 CRMs validated as functional in the assay. The rate of predictive success reveals striking limitations of computational regulatory sequence analysis methods for CRM discovery. Motif-based methods performed no better than predictions based only on sequence conservation. Analysis of the properties of the functional sequences relative to inactive sequences identifies nucleotide sequence composition can be an important characteristic to incorporate in future methods for improved predictive specificity. Muscle-related TFBSs predicted within the functional sequences display greater sequence conservation than non-TFBS flanking regions. Comparison with recent MyoD and histone modification ChIP-Seq data supports the validity of the functional regions
Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets
DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a−/− cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a−/− cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease
Transcription and Chromatin Organization of a Housekeeping Gene Cluster Containing an Integrated β-Globin Locus Control Region
The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced
Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.
Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called episignatures ). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders
Heterochromatin and the molecular mechanisms of 'parent-of-origin' effects in animals.
Twenty five years ago it was proposed that conserved components of constitutive heterochromatin assemble heterochromatinlike complexes in euchromatin and this could provide a general mechanism for regulating heritable (cell-to-cell) changes in gene expressibility. As a special case, differences in the assembly of heterochromatin-like complexes on homologous chromosomes might also regulate the parent-of-origin-dependent gene expression observed in placental mammals. Here, the progress made in the intervening period with emphasis on the role of heterochromatin and heterochromatin-like complexes in parent-of-origin effects in animals is reviewed
A roupa nova do presidente: a politização da imagem pública de Jânio Quadros (1947-1961)
Jânio Quadros public image is often considered deprived of any political meaning either by historians or by collective memory. In most cases he is treated as a demagogue who promoted himself only through his personalism, which could be seen in his public appearances unkempt and with open-necked shirts. Such perspective is deeply related to the way his opponents and enemies represented him in the political debates, and its core has been successful in penetrating the historiography. However in this text I intend to suggest that Jânio's images conveyed an array of political senses during the 1950s and early 1960s, since the beginning of his career till when he disputed presidential elections and ruled Brazil as president. Through his gestures and clothes, among which his Indian slacks deserve more attention, he tried to express his project related to the Christian Democracy and the Independent Foreign Policy. To deal with such hypothesis, the main sources will be the magazines O Cruzeiro and Manchete and others photos and drawings that were considerably widespread at that time.A imagem pública de Jânio Quadros é frequentemente considerada como desprovida de quaisquer significados políticos, seja por historiadores ou pela memória coletiva. Na maioria dos casos, ele é tratado como um demagogo que se promovia apenas através de seu personalismo, exibindo-se em suas aparições públicas despenteado e com camisas abertas no colarinho. Tal perspectiva está profundamente relacionada com a forma pela qual seus oponentes e inimigos o representavam nos debates políticos, e em seu cerne foi bem-sucedida, tendo penetrado na historiografia. Neste texto, porém, pretendo sugerir que, perpassando a década de 1950 e o início da de 1960, desde o início de sua carreira até o momento em que disputou eleições presidenciais e foi o presidente do Brasil, as imagens de Jânio veiculavam um amplo leque de sentidos políticos. Por meio de gestos e roupas, destacando-se aí seus slacks indianos, ele procurou expressar seus projetos relacionados à Democracia Cristã e à Política Externa Independente. Para lidar com essa hipótese, as principais fontes fotográficas e textuais elencadas são as revistas O Cruzeiro e Manchete da época, e outras fotos e ilustrações que então foram bastante difundidas
- …