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A short ORF-encoded transcriptional regulator - PubMed

  • ️Fri Jan 01 2021

. 2021 Jan 26;118(4):e2021943118.

doi: 10.1073/pnas.2021943118.

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A short ORF-encoded transcriptional regulator

Minseob Koh et al. Proc Natl Acad Sci U S A. 2021.

Abstract

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Keywords: expanded genetic code; photo-crosslinking; short open reading frame-encoded peptide; transcriptional regulation.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

A genetically encoded photo-crosslinker identifies cellular interactors of unexplored SEPs. (A) Schematic depicting the strategy used in this work to introduce the photo-crosslinking amino acid AbK (structure within inset circle) into SEP transgenes and identify their protein targets in cells. (B) Heatmap of the relative spectral count enrichment corresponding to protein preys identified from AP-MS experiments with the indicated SEP baits in the presence or absence of UV (mean of three biological replicates).

Fig. 2.
Fig. 2.

SEHBP is a translated 5′ ORF that is conserved in mammals. (A) Schematic depicting the position of the SEHBP and ZNF689 ORFs within transcript NM_138447. (B) Multiple sequence alignment depicting the sequence (Top), conservation (Middle), and consensus (Bottom) of the SEHBP amino acid sequence across the indicated mammalian species. (C) Representative A-site plots (Ribo-seq) from the indicated cell lines with the ORF of SEHBP shown in yellow.

Fig. 3.
Fig. 3.

SEHBP interacts with Histone H2B in cells. (A) Relative spectral counts of the indicated anti-FLAG immunoprecipitated proteins after 48-h expression of SEHBP-AbK-FLAG in HEK293T cells followed by exposure to UV (n = 3; mean ± SD). (B) Representative confocal microscopy-derived images of SEHBP-eGFP localization (green) from HEK293T cells after 48 h of SEHBP-eGFP expression followed by fixation and exposure to Hoechst 33342. (C) Representative Western blotting analysis for the indicated proteins after 48-h expression of SEHBP-FLAG followed by subcellular fractionization. (D) Western blotting for endogenous H2B content after anti-FLAG immunoprecipitation from HEK293T cells expressing SEHBP-FLAG. (E) Western blotting analysis for SEBHP-FLAG content after anti-H2B immunoprecipitation from HEK293T cells expressing SEHBP-FLAG. (F) Sensorgram plot from biolayer interferometry experiments demonstrating the interaction between recombinant H2B and immobilized SEHBP.

Fig. 4.
Fig. 4.

SEHBP modulates transcription at distinct loci. (A) Rank-ordered heat map depicting the log2-fold change of mRNA levels in response to SEHBP overexpression relative to a vector control in RNA-seq experiments using HEK293T cells (three biological replicates). (B) Pie chart depicting the percentage of active transcripts significantly changed by SEHBP overexpression (P < 0.05). (C) Histogram comparing the normalized enrichment scores of all MSigDB gene sets using GSEA of RNA-seq data in response to SEHBP overexpression. Plots depicting the P values of representative up-regulated (D) and down-regulated (E) gene sets from RNA-seq analysis of HEK293T cells expressing SEHBP. (F) Rank-ordered heat map of ChIP-seq derived reads of SEHBP-based enrichment at 8,900 distinct loci (Left) and 1,786 TSSs (Right).

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