Biology subjects

Shetty, S. J.

Publications and source records attributed to Shetty, S. J..

3 recordsLinked to original sources

bioRxiv2026

Microtubule binding protein Togaram1 is required for proper development of mammalian forebrain and neural primary cilia

Proper forebrain development relies on precise spatial and temporal control of early neural stem cell (NSC) proliferation and later neurogenesis. Brain malformations can arise when these processes are defective. Joubert Syndrome (JS) is a neurodevelopmental disorder that is diagnosed by a mid-hindbrain malformation, but often includes forebrain defects such as microcephaly, which are less understood. One gene recently linked to Joubert Syndrome with microcephaly is Togaram1, which encodes a TOG domain microtubule binding protein shown to affect primary cilia. In the embryonic dorsal forebrain, NSCs have primary cilia on their apical membranes that play a role in regulating proliferation and neurogenesis, but how they do this is not well understood. Here we investigate the role of Togaram1 in mammalian forebrain development using a mouse knockout. We find that Togaram1 is crucial for forebrain size, thickness, and morphology. In particular, knockout forebrains have sporadic indentations of the lateral ventricles, and the neuronal layer is thin with gaps and heterotopias. The dorsal forebrain NSCs have increased proliferation and apoptosis. Finally, the primary cilia of Togaram1 knockout NSCs have abnormal morphology and function. This study begins to elucidate the role of Togaram1 in forebrain morphogenesis and the involvement of NSC primary cilia in forebrain malformations.

developmental biology↗

bioRxiv2023

Genome-scale chromatin interaction dynamic measurements for key components of the RNA Pol II general transcription machinery

BackgroundA great deal of work has revealed in structural detail the components of the machinery responsible for mRNA gene transcription initiation. These include the general transcription factors (GTFs), which assemble at promoters along with RNA Polymerase II (Pol II) to form a preinitiation complex (PIC) aided by the activities of cofactors and site-specific transcription factors (TFs). However, less well understood are the in vivo PIC assembly pathways and their kinetics, an understanding of which is vital for determining on a mechanistic level how rates of in vivo RNA synthesis are established and how cofactors and TFs impact them. ResultsWe used competition ChIP to obtain genome-scale estimates of the residence times for five GTFs: TBP, TFIIA, TFIIB, TFIIE and TFIIF in budding yeast. While many GTF-chromatin interactions were short-lived (< 1 min), there were numerous interactions with residence times in the several minutes range. Sets of genes with a shared function also shared similar patterns of GTF kinetic behavior. TFIIE, a GTF that enters the PIC late in the assembly process, had residence times correlated with RNA synthesis rates. ConclusionsThe datasets and results reported here provide kinetic information for most of the Pol II-driven genes in this organism and therefore offer a rich resource for exploring the mechanistic relationships between PIC assembly, gene regulation, and transcription. The relationships between gene function and GTF dynamics suggest that shared sets of TFs tune PIC assembly kinetics to ensure appropriate levels of expression.

biochemistry↗

bioRxiv2017

Second-generation method for analysis of chromatin binding using formaldehyde crosslinking kinetics

Formaldehyde crosslinking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called CLK, which uses time-dependent formaldehyde crosslinking data to extract chromatin binding kinetic parameters. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially impact analyses of CLK data. Here we report biochemical results that better define the properties of formaldehyde crosslinking in budding yeast cells. These results have the potential to inform interpretations of standard chromatin assays including chromatin immunoprecipitation, and the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics using the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine quench conditions. Notably, we find that formaldehyde crosslinking rates can vary dramatically for different protein-DNA interactions in vivo. Some interactions were crosslinked much faster than the time scale for macromolecular interaction, making them suitable for kinetic analysis. For other interactions, we find the crosslinking reaction occurred on the same time scale or slower than binding dynamics; for these it was in some cases possible to compute the in vivo equilibrium-binding constant but not on- and off-rates for binding. Selected TATA-binding protein-promoter interactions displayed dynamic behavior on the minute to several minutes time scale.

biochemistry↗