# Mammalian Proteome Profiling Reveals Readers and Antireaders of Strand‐Symmetric and ‐Asymmetric 5‐Hydroxymethylcytosine‐Modifications in DNA

## TL;DR

This study identifies proteins that recognize specific DNA modifications, revealing how these modifications may regulate gene activity in development and disease.

## Contribution

The study discovers new reader proteins for symmetric and asymmetric 5-hydroxymethylcytosine DNA modifications and their regulatory roles.

## Key findings

- MYC and MAX are identified as sequence-dependent readers of hmC modifications.
- RFX5 discriminates between hmC symmetries in CpG dyads.
- Tissue-specific reader proteins are linked to chromatin regulation and disease.

## Abstract

The cytosine (C) modifications 5‐methylcytosine (mC) and 5‐hydroxymethylcytosine (hmC) are central regulatory elements of mammalian genomes. Both marks occur in double‐stranded DNA in either strand‐symmetric or ‐asymmetric fashion, but it is still poorly understood how this symmetry information is selectively read out by the nuclear proteome as the basis of potential symmetry‐dependent regulation. We report enrichment/proteomics studies with promoter probes being strand‐symmetrically or asymmetrically modified with C, mC, and hmC, enabling comparison of their reader profiles in the same sequence, tissue, and experimental contexts. We identify a high number of tissue‐specific readers for hmC‐modified sequences that fall into distinct, probe‐specific sub‐groups, including members of important transcription factor classes and chromatin regulators. Among them, we discover the master regulators MYC and MAX that play central roles in cell (de)differentiation and cancer progression to read hmC in a sequence‐dependent manner. We also find RFX5, a transcription factor involved in primary MHC class II deficiency, to discriminate between hmC symmetries in CpG dyads. Our findings provide further support for the hypothesis that hmC symmetry information can provide distinct regulatory outputs and provide a resource for studying the molecular mechanisms triggered by symmetric and asymmetric hmC modifications in chromatin regulation during development and disease.

We investigate by proteomics studies how strand‐symmetric and ‐asymmetric cytosine 5‐modifications in DNA are selectively recognized by the nuclear proteome. Using promoter probes with defined modification patterns, we identify tissue‐specific reader proteinsincluding MYC, MAX, and RFX5that discriminate 5‐hydroxymethylcytosine symmetry and sequence context. Our findings provide biochemical support for the existence of symmetry‐dependent regulatory mechanisms of 5‐hydroxymethylcytosine in chromatin control and provide a resource for studying their roles in development and disease.