Robert Tjian

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Regulatory diversity among metazoan co-activator complexes

Transcription is a stepwise process that involves many specialized proteins and protein complexes, all of which must work together to express a given gene in a spatially and temporally regulated manner. An integral step in this regulatory process is carried out by large, multisubunit co-activator complexes, which have diverse roles in transcriptional control. Their diversity and large size allows for many potential regulatory inputs, but how is the versatility and specificity of these co-activator complexes determined?

Distinct conformational states of nuclear receptor–bound CRSP–Med complexes

The human CRSP–Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP–Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP–Med complex. To test this, we conducted a structural analysis of CRSP–Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP–Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 Å) indeed reveals a shared conformational feature that is distinct from other known CRSP– Med structures. Importantly, this nuclear receptor–induced structural shift seems largely dependent on the movement of Med220 within the complex.

Charting Brachyury-mediated developmental pathways during early mouse embryogenesis

Significance The gene-regulatory mechanisms for finely balanced cell-fate determination and morphogenesis during early animal development remain largely elusive. Here, we combine genomic, single-cell imaging and biochemical approaches to chart the molecular pathways mediated by a key developmental regulator—Brachyury. Our results shed light on mechanistic insights into the ultrafine organization of Brachyury-bound enhancers and link Brachyury function to cellular differentiation and housekeeping processes critical for coordinating early mouse embryogenesis. To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator—Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the Tc mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.

A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

TFIID—a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)—is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation. DOI: http://dx.doi.org/10.7554/eLife.08536.001

Visualizing transcription factor dynamics in living cells

Liu and Tjian review how superresolution and live-cell imaging are providing new insights into transcription factor dynamics and genome organization.

Regulation of DNA demethylation by the XPC DNA repair complex in somatic and pluripotent stem cells

Faithful resetting of the epigenetic memory of a somatic cell to a pluripotent state during cellular reprogramming requires DNA methylation to silence somatic gene expression and dynamic DNA demethylation to activate pluripotency gene transcription. The removal of methylated cytosines requires the base excision repair enzyme TDG, but the mechanism by which TDG-dependent DNA demethylation occurs in a rapid and site-specific manner remains unclear. Here we show that the XPC DNA repair complex is a potent accelerator of global and locus-specific DNA demethylation in somatic and pluripotent stem cells. XPC cooperates with TDG genome-wide to stimulate the turnover of essential intermediates by overcoming slow TDG-abasic product dissociation during active DNA demethylation. We further establish that DNA demethylation induced by XPC expression in somatic cells overcomes an early epigenetic barrier in cellular reprogramming and facilitates the generation of more robust induced pluripotent stem cells, characterized by enhanced pluripotency-associated gene expression and self-renewal capacity. Taken together with our previous studies establishing the XPC complex as a transcriptional coactivator, our findings underscore two distinct but complementary mechanisms by which XPC influences gene regulation by coordinating efficient TDG-mediated DNA demethylation along with active transcription during somatic cell reprogramming.

Supporting Biomedical Research: Meeting Challenges and Opportunities at HHMI

The Howard Hughes Medical Institute (HHMI) has spent 60 years experimenting and learning how to support biomedical research effectively. From the time Mr Hughes first began funding research in the 1950s, institute leaders and scientists have found that the most important advances and influential discoveries often come unexpectedly—and that often, the profound “breakthroughs” derive from deep fundamental insights of surprising, sometimes obscure biological systems. HHMI’s experience suggests that scientific research is far from understanding the fundamentals of human biology or physiology and that there are several paths to gaining the necessary knowledge of molecular mechanisms governing disease pathologies. Studying model organisms such as yeast, worms, and flies remains a powerful and productive way to learn new biology. Directly studying human diseases caused by single or small numbers of mutations that allow for in-depth analysis (ie, hemoglobinopathies, familial hypercholesterolemia, and cystic fibrosis) offers another effective path to discovering the molecular basis of disease. In both approaches, success requires a sustained, diverse pipeline of scientists inspired to “follow their nose” and equipped with the best research tools available.