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Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation.

ABSTRACT: Mammalian epidermis provides a permeability barrier between an organism and its environment. Under homeostatic conditions, epidermal cells produce structural proteins, which are cross-linked in an orderly fashion to form a cornified envelope (CE). However, under genetic or environmental stress, specific genes are induced to rapidly build a temporary barrier. Small proline-rich (SPRR) proteins are the primary constituents of the CE. Under stress the entire family of 14 Sprr genes is upregulated. The Sprr genes are clustered within the larger epidermal differentiation complex on mouse chromosome 3, human chromosome 1q21. The clustering of the Sprr genes and their upregulation under stress suggest that these genes may be coordinately regulated. To identify enhancer elements that regulate this stress response activation of the Sprr locus, we utilized bioinformatic tools and classical biochemical dissection. Long-range comparative sequence analysis identified conserved noncoding sequences (CNSs). Clusters of epidermal-specific DNaseI-hypersensitive sites (HSs) mapped to specific CNSs. Increased prevalence of these HSs in barrier-deficient epidermis provides in vivo evidence of the regulation of the Sprr locus by these conserved sequences. Individual components of these HSs were cloned, and one was shown to have strong enhancer activity specific to conditions when the Sprr genes are coordinately upregulated.

SUBMITTER: Martin N

PROVIDER: S-EPMC534667 | biostudies-other | 2004 Dec

REPOSITORIES: biostudies-other

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Project description:Here we focus on identifying conserved elements that are required for Chaserr function, a lncRNA that we recently characterized as being essential for mouse viability (https://doi.org/10.1038/s41467-019-13075-8). Chaserr is highly conserved in different species, and is located in close proximity (<2kb) to the transcription start site of CHD2. It acts in concert with the CHD2 protein to maintain proper Chd2 expression levels. Using the LncLOOM framework we identified AUGG enriched conserved elements which are located in the last exon of mouse Chaserr. In order to identify proteins that potentially bind to these conserved regions, we used in vitro transcription to generate the following biotinylated RNAs; along with their antisense controls: 1) The WT sequence, containing the first 322 nt of the last exon of Chaserr (bases 764–1086 of the NR_037601 isoform). An antisense control fragment was also prepared, labelled as AWT 2) The mutant conserved (MC) sequence, that contained AUGG→UACC mutations in four conserved motifs that were found using LncLOOM. An antisense control fragment was also prepared, labelled as AMC 3) The muatant all (MA) sequence in which all seven of the AUGG sites in the last exon were mutated to UACC. An antisense control fragment was also prepared, labelled as AMA 4) We also included a No Probe Control, which did not contain an RNA fragment. The fragments were incubated with N2a cell lysates followed by RNA-pulldown and Mass Spec analysis. The experiment was performed in triplicate. A total of 938 proteins were found to associate with the WT sequence: 74 of these were enriched ≥3-fold compared to the antisense sequence.We identified 9 proteins that had ≥2-fold higher recovery when using the WT sequence compared to both mutants. Included in these proteins was DHX36, which showed the highest enrichment compared to the mutated sequences. We validated the association of DHX36 with Chaserr through RNA immunoprecipitation experiments.

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Long-range comparison of human and mouse Sprr loci to identify conserved noncoding sequences involved in coordinate regulation.

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