Identification and functional characterization of transcriptional activators in human cells.

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

The N-terminus of Spt16 anchors FACT to MCM2-7 for parental histone recycling.

Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4 and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance.

FACT interacts with Set3 HDAC and fine-tunes GAL1 transcription in response to environmental stimulation.

The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3' end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.

Characterization of a Fiber Bundle-Based Real-Time Ultrasound/Photoacoustic Imaging System and Its In Vivo Functional Imaging Applications.

Photoacoustic (PA) imaging is an attractive technology for imaging biological tissues because it can capture both functional and structural information with satisfactory spatial resolution. Current commercially available PA imaging systems are limited by their bulky size or inflexible user interface. We present a new handheld real-time ultrasound/photoacoustic imaging system (HARP) consisting of a detachable, high-numerical-aperture (NA) fiber bundle-based illumination system integrated with an array-based ultrasound (US) transducer and a data acquisition platform. In this system, different PA probes can be used for different imaging applications by switching the transducers and the corresponding jackets to combine the fiber pads and transducer into a single probe. The intuitive user interface is a completely programmable MATLAB-based platform. In vitro phantom experiments were conducted to test the imaging performance of the developed PA system. Furthermore, we demonstrated (1) in vivo brain vasculature imaging, (2) in vivo imaging of real-time stimulus-evoked cortical hemodynamic changes during forepaw electrical stimulation, and (3) in vivo imaging of real-time cerebral pharmacokinetics in rats using the developed PA system. The overall purpose of this design concept for a customizable US/PA imaging system is to help overcome the diverse challenges faced by medical researchers performing both preclinical and clinical PA studies.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication.

The Histone Chaperone FACT Contributes to DNA Replication-Coupled Nucleosome Assembly.

DNA replication-coupled (RC) nucleosome assembly is mediated by histone chaperones and is fundamental for epigenetic inheritance and maintenance of genomic integrity. The mechanisms that promote this process are only partially understood. Here, we show that the histone chaperone FACT (facilitates chromatin transactions), consisting of Spt16 and Pob3, promotes newly synthesized histone H3-H4 deposition. We describe an allele of Spt16 (spt16-m) that has a defect in binding to H3-H4 and impairs their deposition onto DNA. Consistent with a direct role for FACT in RC nucleosome assembly, spt16-m displays synthetic defects with other histone chaperones associated with this process, CAF-1 and Rtt106. Importantly, we show that FACT physically associates with Rtt106 and that the acetylation of H3K56, a mark on newly synthesized H3, modulates this interaction. Therefore, FACT collaborates with CAF-1 and Rtt106 in RC nucleosome assembly.