Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution.

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Acetylation reprograms MITF target selectivity and residence time.

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.

A TFEB nuclear export signal integrates amino acid supply and glucose availability.

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3ß phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3ß is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin.

Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.

The seasonal sea-ice zone in the glacial Southern Ocean as a carbon sink.

Reduced surface-deep ocean exchange and enhanced nutrient consumption by phytoplankton in the Southern Ocean have been linked to lower glacial atmospheric CO2. However, identification of the biological and physical conditions involved and the related processes remains incomplete. Here we specify Southern Ocean surface-subsurface contrasts using a new tool, the combined oxygen and silicon isotope measurement of diatom and radiolarian opal, in combination with numerical simulations. Our data do not indicate a permanent glacial halocline related to melt water from icebergs. Corroborated by numerical simulations, we find that glacial surface stratification was variable and linked to seasonal sea-ice changes. During glacial spring-summer, the mixed layer was relatively shallow, while deeper mixing occurred during fall-winter, allowing for surface-ocean refueling with nutrients from the deep reservoir, which was potentially richer in nutrients than today. This generated specific carbon and opal export regimes turning the glacial seasonal sea-ice zone into a carbon sink.

Spatial organization of intestinal microbiota in the mouse ascending colon.

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host-microbial interactions.