Lineage-specific transcriptional regulation of DICER by MITF in melanocytes

DICER is a central regulator of microRNA maturation. However, little is known about mechanisms regulating its expression in development or disease. While profiling miRNA expression in differentiating melanocytes, two populations were observed: some upregulated at the pre-miRNA stage, and others upregulated as mature miRNAs (with stable pre-miRNA levels). Conversion of pre-miRNAs to fully processed miRNAs appeared to be dependent upon stimulation of DICER expression--an event found to occur via direct transcriptional targeting of DICER by the melanocyte master transcriptional regulator MITF. MITF binds and activates a conserved regulatory element upstream of DICER's transcriptional start site upon melanocyte differentiation. Targeted KO of DICER is lethal to melanocytes, at least partly via DICER-dependent processing of the pre-miRNA-17 approximately 92 cluster thus targeting BIM, a known proapoptotic regulator of melanocyte survival. These observations highlight a central mechanism underlying lineage-specific miRNA regulation which could exist for other cell types during development.

Rhythmic and clock-dependent chromatin loops regulate circadian gene expression

Jérôme Mermet

In mammals the circadian clock drives daily behavioural and physiological changes that resonate with environmental cues, which can be observed, for example, in the intricate timing of rest during the night and activity during the day in humans. The circadian clock consists of a network of hierarchical clocks in which the central pacemaker is located in the suprachiasmatic nucleus, which is sensitive to external light and propagates time to the peripheral organ-based clocks. Within organs, clocks tick in each individual cell and are molecularly encoded in the genome as a network of dynamic feedback loops. By mediating the expression of the appropriate gene products at the correct moment of the day, the molecular clocks op-timize physiological functions of virtually every cell in our body, allowing us to synchronize with daily fluc-tuations in the environment. In mammalian cells the chromatin organization in the nucleus consists of a topological network, in which chromatin interactions between distal regulatory elements such as enhancers and target genes are essen-tial to regulate gene expression. Important questions remain: is the complex chromatin folding dynamic and if so on which genomic and temporal scales? In fact, dynamic rearrangements of the chromatin have been reported on multiple genomic and time scales, from the entire genome to enhancer-promoter con-tacts, across developmental transitions, cellular differentiation, and transcription cycles. With this per-spective in mind, the circadian oscillator provides a unique model to study the dynamics of genomic inter-actions in the context of fluctuating transcription. Here, we explored chromatin contacts of core-clock and clock-controlled gene promoters in the mouse. We aimed at evaluating the putative dynamics of chromatin contacts, at estimating their conservation across tissues, and at exploring the contribution of the circadian clock to this interaction network. To achieve this goal, we performed 4C-sequencing assays at two circadian time points in the liver and kidney of WT and clock-deficient animals. Overall, we observe that chromatin contacts are largely conserved across circadian time points and genetic backgrounds, and are tissue-specific. Our experiments reveal that the promoters of core-clock and clock-controlled genes contact distal genomic regions containing en-hancer chromatin signatures, suggesting a functional role of these distal sites in target gene regulation. Our data also suggest a clock-driven module of rhythmic genes that are colocalized in the nucleus. However, we also observe dynamic contacts between oscillating gene promoter and enhancer-like ge-nomic regions. In this thesis, we discuss in detail two examples of dynamic chromatin looping that are ob-served at the Cry1 and Gys2 loci. Our data reveal not only a dynamic coupling between the chromatin loop and the transcriptional state but also that the dynamic of chromatin looping depends on a functional clock. Furthermore, genome editing experiments using CRISPR-Cas9 tools reveal that the Cry1 distal site is essential for the transcriptional regulation of Cry1 and contributes to the circadian clock robustness both in-vivo and in cultured cells. These results demonstrate how local chromatin rearrangements take place in a 24-hour time-scale and set chromatin looping between gene promoters and distal regions as a new reg-ulatory layer for circadian gene expression.

EPFL2017

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

Unraveling the KRAB/KAP1 control of transposable elements in pluripotent and somatic cells

Gabriela Ecco

Transposable elements (TEs) are DNA sequences able to change position in the genome, and represent more than 40% of mammalian genetic material. TEs can have positive or detrimental effects on the host, being both important motors of evolution and genomic threats, and are associated with diseases such as cancer and diabetes. Growing evidence indicates the host co-opts TEs for its benefit, with examples such as syncytins â genes important for placental formation that derive from TEs. Given their potential damaging role, the host needs to repress TEs, especially during early embryogenesis. KRAB-containing zinc finger proteins (KRAB-ZFPs) are important regulators of TEs, which they repress with their cofactor KAP1. In stem cells, the KRAB/KAP1 complex irreversibly silence TEs and is believed to be dispensable in adult cells. KRAB-ZFPs constitute one of the largest families of transcriptional regulators encoded by higher vertebrates, but functional information is missing for most of its members. To shed light on this important family of transcription factors, we first developed a large-scale functional screen matching murine TEs with their cognate KRAB-ZFP. The screen identified KRAB-ZFP809 as the ligand of its previously mapped DNA target in the murine leukemia virus genome. Our method further singled out two novel KRAB-ZFPs binding to TE sequences, as confirmed by repression and binding assays. One of them, Gm6871, was identified to regulate LINE-1 elements together with KAP1 in embryonic stem cells. KAP1 regulation followed a chronologically conditioned pattern, repressing elements that entered the mouse genome between 5.6 and 3.8 million years ago, suggestive of an evolutionary arms race between host KRAB-ZFPs and TEs. Secondly, we found the paralogs ZFP932 and Gm15446 to bind overlapping but distinguishable subsets of ERVK (endogenous retrovirus K), to repress these elements in embryonic stem cells, and to regulate secondarily the expression of neighboring genes. Furthermore, we uncovered that these KRAB-ZFPs and KAP1 control TEs in adult tissues, in cell culture and in vivo, where they partner up to modulate cellular genes. Our results establish an efficient screening method to identify KRAB-ZFPs DNA targets, opening the way to functional analyses of this major class of transcriptional repressors. Most importantly, our study strongly suggests that KRAB-ZFP genes are not only part of an evolutionary arms race with TEs, but also participate in the domestication of these elements for the benefit of the host. Given the abundance and high degree of species-specificity of TEs and KRAB-ZFPs, these results have important implications for understanding the biology of higher vertebrates, including humans.