# The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase

## TL;DR

This paper shows that the ASB9-CUL5 ubiquitin ligase can directly ubiquitylate histones H3 and H4, promoting their degradation without needing a helper enzyme.

## Contribution

The study reveals a novel ubiquitylation mechanism by ASB9-CRL5 that does not require the RBR ligase ARIH2.

## Key findings

- ASB9-CRL5 preferentially polyubiquitylates histones H3 and H4.
- The ubiquitylation process does not require the RBR ligase ARIH2.
- ASB9-CRL5 binds histones through electrostatic interactions and targets free histones for degradation.

## Abstract

The E3 ligase substrate receptor ankyrin and suppressor of cytokine signaling box protein 9 (ASB9) was shown to bind over 10 different proteins including metabolic enzymes such as creatine kinase, filament proteins such as vimentin, and histones. In previous work, we characterized the ASB9-Cullin 5 E3 ligase (ASB9-CRL5) ubiquitylation of creatine kinase and showed that ubiquitylation required the ring-between-ring ligase, ARIH2. Here, we characterized the ASB9-CRL5 ubiquitylation of histones and show that histones histone 3 (H3) and histone 4 (H4) are polyubiquitylated by the ASB9-CRL5 whereas histones Histone 2A and Histone 2B are much poorer substrates. Many, but not all lysines in the histones are ubiquitylated suggesting some substrate specificity. Binding experiments show that the ligase-histone interaction is highly electrostatic and the neddylated ASB9-CRL5 binds with the highest affinity. Histones in nucleosomes or in complex with the chaperone Asf1, are not ubiquitylated. Only K48 and K63 polyubiquitin chains were observed, suggesting that the ubiquitylation probably drives histone degradation. The presence of ASB9 in specific cell types correlates with situations in which free histones H3 and H4 need to be degraded. In this work, we demonstrate that the ASB9-CRL5 is the ligase that facilitates degradation of histones H3 and H4. In addition, this work represents the first example of Cullin-5 mediated ubiquitylation that does not require a ring-between-ring “helper” ligase.

•Histones H3 and H4 are bona fide substrates for the ASB9-CRL.•Histones H3 and H4 are preferentially polyubiquitylated by the ASB9-CRL.•Polyubiquitylation of H3 and H4 by the ASB9-CRL does not require the RBR ARIH2.

Histones H3 and H4 are bona fide substrates for the ASB9-CRL.

Histones H3 and H4 are preferentially polyubiquitylated by the ASB9-CRL.

Polyubiquitylation of H3 and H4 by the ASB9-CRL does not require the RBR ARIH2.

The ASB9-CUL5 Ubiquitin Ligase was identified as the E3 ligase that polyubiquitylates extranucleosomal histones H3 and H4 by a novel mechanism not requiring ARIH2. ASB9 is found mainly in testes, where degradation of free histones is linked to spermatoid maturation.