Lim, S. M. | Search bioRxiv

bioRxiv2020

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins

The nuclear position in eukaryotic cells is controlled by a nucleo-cytoskeletal network, with important roles in cell differentiation, division and movement. Forces are transmitted through conserved linker of nucleoskeleton and cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2 giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin network directly as well as through FHOD1, a formin whose major activity is bundling actin. Much of the molecular details of this process remain poorly understood. Here, we report the crystal structure of Nes2G bound to FHOD1. We show that the G-binding domain of FHOD1 is rather a spectrin repeat binding enhancer for the neighboring FH3 domain, possibly establishing a common binding mode among this subclass of formins. The FHOD1-Nes2G complex structure suggests that spectrin repeat binding by FHOD1 is likely not regulated by the DAD helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding spectrin repeat, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement.

biochemistry↗

bioRxiv2020

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

genomics↗