Nuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation are not well understood. Studying how the lncRNA Xist establishes the inactive-X-chromosome (Xi)-compartment, we found that the Xist RNA-binding-proteins PTBP1, MATR3, TDP43, and CELF1 form a condensate to create an Xidomain that can be sustained in the absence of Xist. The E-repeat-sequence of Xist serves a multivalent binding-platform for these proteins. Without the E-repeat, Xist initially coats the Xchromosome during XCI onset but subsequently disperses across the nucleus with loss of gene silencing. Recruitment of PTBP1, MATR3, TDP-43 or CELF1 to DE-Xist rescues these phenotypes, and requires both self-association of MATR3 and TDP-43 and a heterotypic PTBP1-MATR3-interaction. Together, our data reveal that Xist sequesters itself within the Xi-territory and perpetuates gene silencing by seeding a protein-condensate. Our findings uncover an unanticipated mechanism for epigenetic memory and elucidate the interplay between RNA and RNA-bindingproteins in creating compartments for gene regulation. Main textThe function of long non-coding RNAs (lncRNAs) and the mechanisms by which they act remain largely unknown. One of the best studied lncRNAs is Xist, which orchestrates X-chromosome inactivation (XCI) in placental female mammals 1-7 . By spreading across one X-chromosome and mediating chromosome-wide gene silencing, Xist equalizes X-linked gene expression with that of males 8-12 . XCI initiates when Xist is induced on one of the two X-chromosomes in pluripotent cells of the implanting blastocyst, or upon induction of differentiation in embryonic stem cells (ESCs) 4,13 , the latter providing a powerful model for the mechanistic dissection of XCI-initiation.Intriguingly, Xist shapes nuclear organization during XCI-initiation. Xist establishes a transcriptionally silent, intra-chromosomal domain (or compartment) by specifically localizing to the X-chromosome from which it is transcribed and inducing the compaction of the forming inactive X-chromosome (Xi), the enrichment of heterochromatin proteins, the repositioning of silenced genes into the center of the Xi, and the exclusion of active transcriptional regulators, such as RNA polymerase II 1,2,14-20 . Yet, the mechanisms that drive and maintain the Xist RNA within a spatially confined region to establish this Xi-domain remain unclear.6 the E-repeat occurred on the 129 allele, which also harbors 11 copies of an MS2-RNA tag within Xist 15 , yielding the X129 Xist DE, MS2 XCas Xist WT genotype (referred to as DE ESCs below) ( Fig. 2a and Extended Data Fig. 5a-d). We ensured that DE ESCs maintained two X-chromosomes, and differentiated normally, as judged by morphological changes and loss of NANOG expression (Extended Data Fig. 5e-g). When transcribed from the X129 Xist DE, MS2 allele, Xist exon 6 was spliced to a cryptic site within exon 7 to generate an RNA missing specifically the E-repeat (Extended Data Fig. 6).RNA FISH over ...
Cortical interneurons are indispensable for proper function of neocortical circuits. Changes in interneuron development and function are implicated in human disorders, such as autism spectrum disorder and epilepsy. In order to understand human-specific features of cortical development as well as the origins of neurodevelopmental disorders it is crucial to identify the molecular programs underlying human interneuron development and subtype specification. Recent studies have explored gene expression programs underlying mouse interneuron specification and maturation. We applied single-cell RNA sequencing to samples of second trimester human ganglionic eminence and developing cortex to identify molecularly defined subtypes of human interneuron progenitors and immature interneurons. In addition, we integrated this data from the developing human ganglionic eminences and neocortex with single-nucleus RNA-seq of adult cortical interneurons in order to elucidate dynamic molecular changes associated with commitment of progenitors and immature interneurons to mature interneuron subtypes. By comparing our data with published mouse single-cell genomic data, we discover a number of divergent gene expression programs that distinguish human interneuron progenitors from mouse. Moreover, we find that a number of transcription factors expressed during prenatal development become restricted to adult interneuron subtypes in the human but not the mouse, and these adult interneurons express species- and lineage-specific cell adhesion and synaptic genes. Therefore, our study highlights that despite the similarity of main principles of cortical interneuron development and lineage commitment between mouse and human, human interneuron genesis and subtype specification is guided by species-specific gene programs, contributing to human-specific features of cortical inhibitory interneurons.
Cortical function critically depends on inhibitory/excitatory balance. Cortical inhibitory interneurons (cINs) are born in the ventral forebrain. After completing their migration into cortex, their final numbers are adjustedduring a period of postnatal development -by programmed cell death. The mechanisms that regulate cIN elimination remains controversial. Here we show that genes in the protocadherin (Pcdh)-γ gene cluster, but not in the Pcdh-α or Pcdh-β clusters, are required for the survival of cINs through a BAX-dependent mechanism. Surprisingly, the physiological and morphological properties of Pcdh-γ deficient and wild type cINs during cIN cell death were indistinguishable. Co-transplantation of wild type and Pcdh-γ deficient interneuron precursor cells demonstrate that: 1) the number of mutant cINs eliminated was much higher than that of wild type cells, but the proportion of mutant or WT cells undergoing cell death was not affected by their density; 2) the presence of mutant cINs increases cell death among wild-type counterparts, and 3) cIN survival is dependent on the expression of Pcdh-γ C3, C4, and C5. We conclude that Pcdh-γ, and specifically γC3, γC4, and γC5, play a critical role in regulating cIN survival during the endogenous period of programmed cIN death. SignificanceInhibitory cortical interneurons (cIN) in the cerebral cortex originate from the ventral embryonic forebrain. After a long migration, they come together with local excitatory neurons to form cortical circuits. These circuits are responsible for higher brain functions, and the improper balance of excitation/inhibition in the cortex can result in mental diseases. Therefore, an understanding of how the final number of cINs is determined is both biologically and, likely, therapeutically significant. Here we show that cell surface homophilic binding proteins belonging to the clustered protocadherin gene family, specifically three isoforms in the Pcdh-γ cluster, play a key role in the regulation cIN programmed cell death. Co-transplantation of mutant and wild-type cINs shows that Pcdh-γ genes have cell-autonomous and non-cell autonomous roles in the regulation of cIN cell death. This work will help identify the molecular mechanisms and cell-cell interactions that determine how the proper ratio of excitatory to inhibitory neurons is determined in the cerebral cortex.
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