In:
Science, American Association for the Advancement of Science (AAAS), Vol. 377, No. 6601 ( 2022-07)
Abstract:
Despite an ever-expanding catalog of noncoding elements that are implicated in the control of mammalian gene expression, how the regulatory input from multiple elements is integrated across a genomic neighborhood has remained largely unclear. This challenge is exemplified at Hox clusters (~100 to 200 kb), which contain genes that specify positional identity along the anterior-posterior axis of the developing embryo. In response to developmental morphogens such as retinoic acid (RA), the HoxA cluster splits into an active ( Hoxa1–5 ) and inactive domain ( Hoxa6–13 ) at the level of gene expression and chromatin. Although distal enhancers, intracluster transcription factor binding, and topological organization have emerged as the major regulatory modules in establishing this expression pattern, their relative contributions remain elusive. RATIONALE Despite the advent of a vast suite of genome editing tools, it has remained challenging to simultaneously manipulate multiple regulatory elements across large genomic windows to deconvolve their relative contributions. Taking inspiration from the bottom-up approaches of synthetic biology and biochemical reconstitution, we developed “synthetic regulatory reconstitution” as a framework for the study of gene regulation to address this gap. The synthesis of large DNA constructs ( 〉 100 kb) permits any combination of complex modifications to be made, at the scale required to probe regulation across a native genomic neighborhood. We fabricated HoxA cluster variants that encode various combinations of the previously identified regulatory modules and integrated them into an ectopic location in the mouse genome. This enabled us to directly test the independent ability of these variant ectopic clusters to reconstitute distinct aspects of HoxA regulation. RESULTS We harnessed the efficient homologous recombination machinery in yeast to construct four rat HoxA variants (130 to 170 kb) and delivered them at single copy to the housekeeping Hprt1 locus of mouse embryonic stem cells. Upon RA-induced differentiation, an ectopic HoxA cluster lacking distal enhancers ( SynHoxA ) induced both the appropriate subset of HoxA genes and the corresponding chromatin boundary. The presence of distal enhancers ( Enhancers+SynHoxA ) increased transcription levels, especially at early time points. Further, both SynHoxA and Enhancers+SynHoxA reorganized into active and inactive topological domains upon differentiation, mirroring the endogenous organization. The mutation of just four retinoic acid response elements (RAREs) present in SynHoxA ( RARE ∆) almost completely eliminated any response of the ectopic cluster to RA, at the levels of both gene expression and chromatin reorganization. The addition of distal enhancers to RARE ∆ could not fully rescue this loss of gene expression phenotype. CONCLUSION Our data suggest that at HoxA , the primary module of active gene and chromatin boundary specification in response to RA is through the presence of internal transcription factor binding sites. Distal enhancers are dispensable for the specification of active genes but synergize with intracluster activator binding to boost the amount of transcription. Therefore, mammalian Hox clusters contain all the regulatory information that is necessary to convert a morphogenetic signal into a stable transcriptional, epigenetic, and topological state. This study showcases the power of synthetic regulatory reconstitution, a generalizable platform for the dissection of gene regulation at other loci in complex genomes. Bottom-up construction of variant mammalian genomic loci at the 〉 100-kb scale. Making multiple edits on the same allele over large genomic windows has remained challenging in mammalian cells. Variant loci with an arbitrary number of changes can be constructed in yeast using synthetic DNA and site-specifically integrated into the genome of mammalian cells to study their behavior. BAC, bacterial artificial chromosome.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abk2820
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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