Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties

Inferring condition-specific transcription factor function from DNA binding and gene expression data

Numerous genomic and proteomic datasets are permitting the elucidation of transcriptional regulatory networks in the yeast Saccharomyces cerevisiae. However, predicting the condition dependence of regulatory network interactions has been challenging, because most protein–DNA interactions identified in vivo are from assays performed in one or a few cellular states. Here, we present a novel method to predict the condition-specific functions of S. cerevisiae transcription factors (TFs) by integrating 1327 microarray gene expression data sets and either comprehensive TF binding site data from protein binding microarrays (PBMs) or in silico motif data. Importantly, our method does not impose arbitrary thresholds for calling target regions ‘bound' or genes ‘differentially expressed', but rather allows all the information derived from a TF binding or gene expression experiment to be considered. We show that this method can identify environmental, physical, and genetic interactions, as well as distinct sets of genes that might be activated or repressed by a single TF under particular conditions. This approach can be used to suggest conditions for directed in vivo experimentation and to predict TF function

Iterative Correction of Hi-C Data Reveals Hallmarks of Chromosome Organization

Extracting biologically meaningful information from chromosomal interactions obtained with genome-wide chromosome conformation capture (3C) analyses requires elimination of systematic biases. We present a pipeline that integrates a strategy for mapping of sequencing reads and a data-driven method for iterative correction of biases, yielding genome-wide maps of relative contact probabilities. We validate ICE (Iterative Correction and Eigenvector decomposition) on published Hi-C data, and demonstrate that eigenvector decomposition of the obtained maps provides insights into local chromatin states, global patterns of chromosomal interactions, and the conserved organization of human and mouse chromosomes

Correlated alterations in genome organization, histone methylation, and DNA-lamin A/C interactions in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease that is frequently caused by a de novo point mutation at position 1824 in LMNA. This mutation activates a cryptic splice donor site in exon 11, and leads to an in-frame deletion within the prelamin A mRNA and the production of a dominant negative lamin A protein, known as progerin. Here we show that primary HGPS skin fibroblasts experience genome-wide correlated alterations in patterns of H3K27me3 deposition, DNA-lamin A/C associations, and, at late passages, genome-wide loss of spatial compartmentalization of active and inactive chromatin domains. We further demonstrate that the H3K27me3 changes associate with gene expression alterations in HGPS cells. Our results support a model that the accumulation of progerin in the nuclear lamina leads to altered H3K27me3 marks in heterochromatin, possibly through the down-regulation of EZH2, and disrupts heterochromatin-lamina interactions. These changes may result in transcriptional misregulation and eventually trigger the global loss of spatial chromatin compartmentalization in late passage HGPS fibroblasts

Cohesin-based chromatin interactions enable regulated gene expression within pre-existing architectural compartments

Chromosome conformation capture approaches have shown that interphase chromatin is partitioned into spatially segregated Mb-sized compartments and sub-Mb-sized topological domains. This compartmentalization is thought to facilitate the matching of genes and regulatory elements, but its precise function and mechanistic basis remain unknown. Cohesin controls chromosome topology to enable DNA repair and chromosome segregation in cycling cells. In addition, cohesin associates with active enhancers and promoters and with CTCF to form long-range interactions important for gene regulation. Although these findings suggest an important role for cohesin in genome organization, this role has not been assessed on a global scale. Unexpectedly, we find that architectural compartments are maintained in non-cycling mouse thymocytes after genetic depletion of cohesin in vivo. Cohesin was however required for specific long-range interactions within compartments where cohesin-regulated genes reside. Cohesin depletion diminished interactions between cohesin-bound sites, while alternative interactions between chromatin features associated with transcriptional activation and repression became more prominent, with corresponding changes in gene expression. Our findings indicate that cohesin-mediated long-range interactions facilitate discrete gene expression states within pre-existing chromosomal compartments

Chromatin interaction analysis reveals changes in small chromosome and telomere clustering between epithelial and breast cancer cells

BACKGROUND: Higher-order chromatin structure is often perturbed in cancer and other pathological states. Although several genetic and epigenetic differences have been charted between normal and breast cancer tissues, changes in higher-order chromatin organization during tumorigenesis have not been fully explored. To probe the differences in higher-order chromatin structure between mammary epithelial and breast cancer cells, we performed Hi-C analysis on MCF-10A mammary epithelial and MCF-7 breast cancer cell lines. RESULTS: Our studies reveal that the small, gene-rich chromosomes chr16 through chr22 in the MCF-7 breast cancer genome display decreased interaction frequency with each other compared to the inter-chromosomal interaction frequency in the MCF-10A epithelial cells. Interestingly, this finding is associated with a higher occurrence of open compartments on chr16-22 in MCF-7 cells. Pathway analysis of the MCF-7 up-regulated genes located in altered compartment regions on chr16-22 reveals pathways related to repression of WNT signaling. There are also differences in intra-chromosomal interactions between the cell lines; telomeric and sub-telomeric regions in the MCF-10A cells display more frequent interactions than are observed in the MCF-7 cells. CONCLUSIONS: We show evidence of an intricate relationship between chromosomal organization and gene expression between epithelial and breast cancer cells. Importantly, this work provides a genome-wide view of higher-order chromatin dynamics and a resource for studying higher-order chromatin interactions in two cell lines commonly used to study the progression of breast cancer

Condition-specific transcription factor binding in yeast and human

The coordinated regulation of gene expression in a condition- and cell type-specific fashion is fundamental to all organisms and vital to human development and homeostasis. Genome-wide measurements of differential gene expression and transcription factor (TF) binding in vivo have provided important information about how such condition-specific gene regulation is accomplished. However, the condition-specific functions of many TFs, even in the extensively studied unicellular yeast Saccharomyces cerevisiae , remain uncharacterized, and even locating potential regulatory regions in the larger genomes of multicellular organisms presents a formidable challenge. Here, I present work predicting and analyzing condition-specific TF-mediated gene regulation in yeast and exploring the importance of distal regulatory regions in human gene regulation. I present an algorithm, CRACR, to predict condition-specific functions and target genes of yeast TFs by integrating comprehensive in vitro TF binding specificity data from protein binding microarrays (PBMs) with gene expression data. With CRACR, I predicted novel target genes and condition-specific functions of both poorly annotated and well-characterized factors in a set of 89 yeast TFs. Several of these target genes and functions were experimentally verified. By comparing PBM data with ChIP-chip in vivo TF binding data, I inferred which in vivo bound regions are direct targets of TFs, and investigated yeast TF preferences for single sites or homotypic clusters of binding sites in vivo. Integrating other genomic datasets, I found that biological functions and regulatory mechanisms of TFs are sometimes shared within DNA binding domain structural classes. To further our understanding of regulatory regions in larger genomes, I analyzed potential cases of distal regulation in human muscle differentiation. I observed myogenic TF binding at predicted cis-regulatory modules (CRMs) distant from gene promoters. With the chromosome conformation capture (3C) method, I found differentiation-specific interactions between two of these distant CRMs and the PDLIM3 and ACTA1 gene promoters. I describe experiments currently underway to characterize the genome-wide interactions of these distal regulatory regions. In the future, combining approaches developed in yeast for detailed analysis of condition-specific TF binding with newly discovered distal regulatory regions in human will lead to a better understanding of gene regulatory mechanisms across organisms

Translocation mapping exposes the risky lifestyle of B cells

Recurrent chromosomal translocations can drive oncogenesis, but how they form has remained elusive. Now, Chiarle et al. (2011) and Klein et al. (2011) characterize the genome-wide spectrum of translocations that form from a single double-stranded break, revealing that specific loci have an intrinsic predisposition for frequent chromosomal rearrangements