Biology subjects

Crocker, O. J.

Publications and source records attributed to Crocker, O. J..

4 recordsLinked to original sources

bioRxiv2025

A human-specific regulatory mechanism revealed in a preimplantation model

Stem cell-based human embryo models offer a unique opportunity for functional studies of the human-specific features of development. Here, we genetically and epigenetically manipulate human blastoids, a 3D embryo model of the blastocyst, to investigate the functional impact of HERVK LTR5Hs, a hominoid-specific endogenous retrovirus, on preimplantation development. We uncover a pervasive cis-regulatory contribution of LTR5Hs elements to the hominoid-specific diversification of the blastoids epiblast transcriptome. Many of the nearly 700 LTR5Hs genomic insertions in the human genome are unique to our own species. We show that at least one such human-specific LTR5Hs element is essential for the blastoid-forming potential via enhancing expression of the primate-specific ZNF729 gene, encoding a KRAB zinc finger protein. ZNF729 binds G/C-rich sequences, extremely abundant at gene promoters associated with basic cellular functions, such cell proliferation and metabolism. Surprisingly, despite mediating recruitment of TRIM28, at many of these promoters ZNF729 acts as a transcriptional activator. Together, our results illustrate how recently emerged transposable elements and genes can confer developmentally essential functions in humans.

developmental biology↗

bioRxiv2025

tRNA-derived RNA processing in sperm transmits non-genetically inherited phenotypes to offspring in C. elegans

The environment encountered by an organism can modulate epigenetic information in gametes to transmit non-genetically inherited phenotypes to offspring. In mouse models, the diet of males regulates specific tRNA-derived RNAs (tDRs) in sperm. After fertilization, tDRs regulate embryonic gene expression and generate metabolic phenotypes in adult offspring through uncharacterized changes during development. Here we demonstrate that in C. elegans, tDRs also accumulate in sperm and can similarly transmit epigenetically inherited phenotypes to offspring. We identify the RNaseT2 enzyme, rnst-2, as a regulator of C. elegans tDR accumulation. RNST- 2 processes or degrades tRNA-halves, leading to short <30 nt fragments. This rnst-2 dependent regulation of tDR length distribution modulates specific tDRs in sperm which, after fertilization, regulate early embryonic and developmental gene expression, leading to adaptive phenotypes in progeny. Our findings establish tDRs as a deeply conserved carrier of intergenerational epigenetic information and position the worm as a model for dissecting paternal non-genetic inheritance mechanistically.

developmental biology↗

bioRxiv2025

Thermodynamic principles link in vitro transcription factor affinities to single-molecule chromatin states in cells

The molecular details governing transcription factor (TF) binding and the formation of accessible chromatin are not yet quantitatively understood - including how sequence context modulates affinity, how TFs search DNA, the kinetics of TF occupancy, and how motif grammars coordinate binding. To resolve these questions for a human TF, erythroid Kruppel-like factor (eKLF/KLF1), we quantitatively compare, in high throughput, in vitro TF binding rates and affinities with in vivo single molecule TF and nucleosome occupancies across engineered DNA sequences. We find that 40-fold flanking sequence effects on affinity are consistent with distal flanks tuning TF search parameters and captured by a linear energy model. Motif recognition probability, rather than time in the bound state, drives affinity changes, and in vitro and in nuclei measurements exhibit consistent, minutes-long TF residence times. Finally, pairing in vitro biophysical parameters with thermodynamic models accurately predicts in vivo single-molecule chromatin states for unseen motif grammars. HighlightsO_LIKLF1:DNA binding is consistent with a three state binding model wherein the probability of recognizing the motif from a nonspecifically-bound state drives affinity. C_LIO_LISubstantial effects from proximal and distal motif-flanking sequence on KLF1 binding affinity are captured by an extended PWM model. C_LIO_LIKLF minutes-long residence time inferred from single-molecule footprinting in nuclei is consistent with in vitro measurement. C_LIO_LIIn vitro binding energies combined with thermodynamic models predict in vivo, single-molecule chromatin configurations across motif grammars and sequence contexts. C_LI

biophysics↗

bioRxiv2024

Long range regulation of transcription scales with genomic distance in a gene specific manner

While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

molecular biology↗