Biology subjects

Bulajic, M.

Publications and source records attributed to Bulajic, M..

4 recordsLinked to original sources

bioRxiv2021

PRC1 Sustains the Memory of Neuronal Fate Independent of PRC2 Function

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We unexpectedly found that PRC2 is dispensable to preserve the morphogen-induced positional fates of spinal motor neurons (MNs), while PRC1 is essential for the specification of segmentally-restricted subtypes. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox networks by derepressed caudal Hox genes. These results indicate that PRC1 can function independently of de novo PRC2-dependent histone methylation to maintain chromatin topology and transcriptional memory at the time of neuronal differentiation.

neuroscience↗

bioRxiv2021

Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce synthetic regulatory reconstitution, a novel conceptual framework for studying gene regulation and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes. One-Sentence SummaryReconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.

synthetic biology↗

bioRxiv2019

Hox binding specificity is directed by DNA sequence preferences and differential abilities to engage inaccessible chromatin.

While Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central, and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aim to address how similar HOX TFs diverge to induce different positional identities. We studied HOX TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We find diversity in the genomic binding profiles of different HOX TFs, even among the posterior group paralogs that share similar DNA binding domains. These differences in genomic binding are explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 has a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior HOX TFs to bind to previously inaccessible chromatin drive patterning diversification.

developmental biology↗

bioRxiv2018

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells. Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized "dark matter" containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context, de novo design, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline for de novo assembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

genetics↗