Chromatin topology and the timing of enhancer function at the HoxD locus

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

A conserved transcriptional enhancer that specifies Tyrp1 expression to melanocytes

Friedrich Beermann

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

2006

Studies on KRAB/KAP1-Mediated Transcriptional Repression

Anna Claire Groner

The sequencing of the human genome revealed that the C2H2-zinc finger proteins (ZFPs) are the largest family of human transcription factors. Around 300 members of this family contain, in addition to their zinc finger motifs, a KRAB domain that has been implicated in transcriptional repression. It is thought that KRAB-ZFPs mediate this effect through the recruitment of the corepressor KAP1, which induces facultative heterochromatin through the activity of several associated chromatin-modifying factors. Our studies focused on the elucidation of the molecular mechanisms of transcriptional repression induced by KRAB-ZFPs and KAP1. For this, we made use of the conditional binding activity of the KRAB-containing repressor tTRKRAB. This ectopic protein serves as a paradigm for endogenous KRAB-ZFP functions, as it depends on cellular KAP1 for transcriptional repression. tTRKRAB only binds to heterologous TetO sites in the absence of doxycycline, thus making repressor activity drug-regulated. When we targeted the repressor to euchromatic sites modified with TetO, we found that it induced increased levels of H3K9me3 and HP1β at the genes it bound to. We also monitored the effect of repressor binding on transcription by assessing the activity of TetO-containing reporter genes that were driven by endogenous promoters and that were initially used to create euchromatic tTRKRAB binding sites. This analysis revealed that KRAB/KAP1 mediated long-range heterochromatin spreading, which only resulted in transcriptional repression if repressive marks reached the promoter and impaired transcriptional initiation. The efficiency of this process was highest in an environment of very strong gene activity, as indicated by the levels of active histone modifications. We found that KRAB-ZFP genes themselves are probably targets of KRAB/KAP1-mediated repression and elucidated their transcriptional regulation and chromatin structure. KAP1 was localized to the promoter and to the 3' end of KRAB-ZFP genes, suggesting self-regulatory loops. However, only KAP1 localized to the 3' end of KRAB-ZFP genes mediated increased levels of H3K9me3 and HP1β along the gene body, without affecting the promoter. Therefore, KAP1 is involved in controlling the spread of heterochromatin at these endogenous sites, largely without regulating transcription. The function of this heterochromatic chromatin landscape may lie in the protection from chromosomal rearrangements between highly homologous KRAB-ZFP genes. Despite this repressive structure, KRAB-ZFP genes are still expressed, as high levels of transcription-associated H3K36me3 accompany H3K9me3 throughout the gene bodies. Other projects described in this thesis include the elucidation of the role of KAP1 in behavior and the assessment of KRAB/KAP1-mediated repression in the context of episomal structures. Both of these areas are of scientific and biomedical interest and address basic principles of the molecular mechanisms of KRAB/KAP1-mediated transcriptional repression.

EPFL2011

Predicting transcription factor site occupancy using DNA sequence intrinsic and cell-type specific chromatin features

Philipp Bucher, Sunil Kumar

Background: Understanding the mechanisms by which transcription factors (TF) are recruited to their physiological target sites is crucial for understanding gene regulation. DNA sequence intrinsic features such as predicted binding affinity are often not very effective in predicting in vivo site occupancy and in any case could not explain cell-type specific binding events. Recent reports show that chromatin accessibility, nucleosome occupancy and specific histone post-translational modifications greatly influence TF site occupancy in vivo. In this work, we use machine-learning methods to build predictive models and assess the relative importance of different sequence-intrinsic and chromatin features in the TF-to-target-site recruitment process. Methods: Our study primarily relies on recent data published by the ENCODE consortium. Five dissimilar TFs assayed in multiple cell-types were selected as examples: CTCF, JunD, REST, GABP and USF2. We used two types of candidate target sites: (a) predicted sites obtained by scanning the whole genome with a position weight matrix, and (b) cell-type specific peak lists provided by ENCODE. Quantitative in vivo occupancy levels in different cell-types were based on ChIP-seq data for the corresponding TFs. In parallel, we computed a number of associated sequence-intrinsic and experimental features (histone modification, DNase I hypersensitivity, etc.) for each site. Machine learning algorithms were then used in a binary classification and regression framework to predict site occupancy and binding strength, for the purpose of assessing the relative importance of different contextual features. Results: We observed striking differences in the feature importance rankings between the five factors tested. PWM-scores were amongst the most important features only for CTCF and REST but of little value for JunD and USF2. Chromatin accessibility and active histone marks are potent predictors for all factors except REST. Structural DNA parameters, repressive and gene body associated histone marks are generally of little or no predictive value. Conclusions: We define a general and extensible computational framework for analyzing the importance of various DNA-intrinsic and chromatin-associated features in determining cell-type specific TF binding to target sites. The application of our methodology to ENCODE data has led to new insights on transcription regulatory processes and may serve as example for future studies encompassing even larger datasets.