Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression–regulating NSL complexes

Hieu T Van,Peter R Harkins,Avni Patel,Abhinav K Jain,Yue Lu,Mark T Bedford,Margarida A Santos

doi:10.1016/j.jbc.2022.101588

Hieu T Van, Peter R Harkins + Show 5 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101588

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Abstract

The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20–PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states.

Highlights

Transcription is a complex process that involves several layers of fine-tuned regulation ranging from chromatin remodeling and histone modifications to RNA processing and export [1]
PHF20 and PHF20Like 1 (PHF20L1) have long been known to complex with NSL, and the assumed models depict both proteins within the same NSL complex [13,16,17]
We further confirmed the MS data by performing IP for HA in 3xHA-PHF20 and 3xHA-PHF20L1 U2OS cells (Supplemental Figure 1A); western blot for PHF20, PHF20L1 and other subunits of the NSL complex such as KAT8, WDR5 and KANSL3 shows that PHF20 and PHF20L1 associate separately with the NSL complex (Figure 1A)

Summary

Introduction

Transcription is a complex process that involves several layers of fine-tuned regulation ranging from chromatin remodeling and histone modifications to RNA processing and export [1]. PHF20 deficiency in mice results in perinatal lethality and various defects in the skeletal and hematopoietic systems; intriguingly, the loss of PHF20 results in decreased gene expression but the levels of H4K16ac remain unaltered [24] This agrees with the recent data demonstrating that the main catalytic activity of the NSL complex is not H4K16ac [15]. We define which domains in each protein are required for the interaction with NSL and the binding to the specific chromatin locations Both PHF20 and PHF20L1 bind to highly expressed genes/housekeeping genes; yet deletion of either or both does not abrogate gene expression at the identified target genes nor the recruitment of NSL to the Journal Pre-proof promoters of those genes, posing the possibility of an alternative compensatory mechanism that sustains the transcription of genes required for crucial cellular functions

Results

Discussion

Experimental procedures

C PHF20L1