CENP-B binds hairpin motifs in chromosome arms influencing gene expression

Lillian Wu, Karen A Lane, Reyhan Muhammad, Alison Harrod, Catherine Naughton, Hefei Wang, Andrea Musacchio, Nick Gilbert, Claudio Alfieri, Jessica A Downs

Posted on: 4 June 2026

Preprint posted on 13 April 2026

CENP-B escapes the centromere and plays a surprising role in gene regulation

Selected by Pierre Caron

Categories: cell biology, genetics, molecular biology

Background

Centromeres are specialized regions of chromosomes that play an essential role during cell division. They serve as the platform for kinetochore assembly, a large protein complex that ensures proper chromosome segregation by attaching chromosomes to spindle microtubules.

At the molecular level, human centromeres are built on long arrays of repetitive α-satellite DNA, organized into higher-order repeats. However, centromere identity is not solely encoded by DNA sequence. Instead, it is primarily defined by a unique chromatin environment enriched in the histone H3 variant CENP-A, which acts as an epigenetic mark specifying where the kinetochore assembles ^1,2.

Surrounding chromatin marked by CENP-A is a network of proteins collectively known as the Constitutive Centromere-Associated Network (CCAN), which stabilizes kinetochore structure and function. One of its best-characterized components is CENP-B, a DNA-binding protein that recognizes specific sequences known as “CENP-B boxes” within α-satellite DNA ^1,2. Historically, CENP-B has been viewed as a centromere-specific factor, yet its functional importance has long been paradoxical: CENP-B knockout mice are viable, and centromeres naturally lacking CENP-B binding sites remain functional under normal conditions. This raises an intriguing possibility: could CENP-B have functions beyond the centromere?

Figure 1. Simplified view of centromeric chromatin and CENP-B binding. Schematic representation of a chromosome highlighting the centromere region. The centromere is composed of CENP-A–containing nucleosomes (green), which define centromere identity, and is flanked by pericentromeric chromatin enriched in canonical H3 nucleosomes (blue). CENP-B (pink) binds specific DNA motifs (CENP-B boxes) within α-satellite repeats, contributing to centromere organization.

In this preprint ³, Wu and colleagues explore this question and uncover an unexpected role for CENP-B outside of centromeres. By combining genome-wide mapping approaches with transcriptional analyses, and by using mutant versions of CENP-B to probe its functional domains, the authors reveal that CENP-B binds to non-centromeric regions and contributes to the regulation of gene expression.

Key findings

CENP-B binds widely outside centromeric regions

Using CUT&RUN (a method for mapping of protein-DNA interactions in vivo), the authors could show that CENP-B is not restricted to centromeric α -satellite DNA but also associates with numerous sites along the chromosome arms. Surprisingly, this binding did not rely on the canonical CENP-B box motif. Instead, non-centromeric CENP-B (nc-CENP-B) binding sites were enriched in repetitive DNA elements, often containing CCAAT motifs and sequences capable of forming secondary structures. These regions also coincided with areas of negative supercoiling, likely generated by active transcription. Consistent with this, the authors demonstrated that CENP-B can directly bind to DNA structures such as hairpins in vitro, suggesting that DNA topology and structure, rather than sequence alone, contribute to its recruitment.

Non-centromeric CENP-B is linked to transcriptionally active regions

Interestingly, nc-CENP-B binding sites were frequently located near promoters, particularly those with divergent transcription. These regions are typically associated with open chromatin and active RNA polymerase II activity.

Despite this localization, CENP-B did not behave like a classical transcription factor. Indeed, gene expression changes observed upon CENP-B depletion did not correlate strongly with its direct binding at promoters. This suggests that CENP-B regulates gene expression through a more indirect mechanism.

CENP-B constrains histone gene expression

To explore the functional consequences of CENP-B binding, the authors performed RNA-seq following CENP-B depletion. While only modest changes were observed at the level of individual genes, a striking pattern emerged when focusing on histone gene clusters.

CENP-B depletion led to increased expression of most histone genes, particularly outside of S phase, when histone expression is normally tightly regulated.

These results suggest that CENP-B normally acts to restrain histone gene expression, preventing inappropriate transcription outside of the replication phase.

Figure 2. Non-centromeric binding of CENP-B and genomic distribution of its targets (panels from Wu et al. ³). (A) Genome browser view showing CENP-B occupancy (CUT&RUN, purple) across a representative genomic region in RPE1 cells, alongside IgG controls. Consensus CENP-B peaks are indicated and overlap with regions of open chromatin detected by ATAC-seq (orange), suggesting that non-centromeric CENP-B binding occurs in accessible genomic regions. (B) Genomic distribution of non-centromeric CENP-B binding sites. The majority of peaks localize to promoter regions, with additional enrichment in intronic and intergenic regions.

A potential role in higher-order chromatin organization

How does CENP-B regulate gene expression if not through direct promoter activity?

The authors propose a compelling model in which CENP-B contributes to higher-order chromatin organization. At centromeres, CENP-B is known to mediate DNA looping. Extending this idea, the authors suggest that nc-CENP-B may organize chromosome arm regions into functional domains that are either permissive or restrictive for transcription.

This model is particularly appealing in the context of histone gene regulation. Histone genes cluster into specialized nuclear structures known as Histone Locus Bodies (HLBs), which assemble during S phase and disassemble afterward. The authors speculate that CENP-B may help coordinate the structural reorganization of these domains, thereby contributing to the temporal control of gene expression.

What I like about this preprint

I really enjoyed reading this preprint because it challenges the long-standing view of CENP-B as a strictly centromeric protein. Instead, the authors reveal a broader and quite unexpected role, suggesting that CENP-B can act beyond centromeres and influence chromatin organization.

What makes this study particularly compelling is the combination of approaches. By bringing together genome-wide mapping, transcriptional analyses, and experiments using CENP-B mutants, the authors move beyond description and start to get at how CENP-B actually works.

Another strong point is the conceptual shift. Rather than behaving like a classical transcription factor, CENP-B seems to regulate gene expression more indirectly, likely through effects on chromatin structure. This is especially interesting in the context of histone genes, where both timing and spatial organization are tightly controlled.

References

Gamba, R. & Fachinetti, D. From evolution to function: Two sides of the same CENP-B coin? Exp. Cell Res. 390, 111959 (2020).
Jaggi, K. E., Hoyt, S. J., O’Neill, R. J. & Sullivan, B. A. A genomic and epigenomic view of human centromeres. Nat. Rev. Genet. 27, 371–384 (2026).
Wu, L. et al. CENP-B binds hairpin motifs in chromosome arms influencing gene expression. Preprint at https://doi.org/10.64898/2026.04.10.717743 (2026).

Tags: cenp-b, chromatin regulation, human cell, transcription

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Author's response

Jessica A Downs shared

Questions for the authors:

Q1 : Do you think CENP-B could have a broader impact on 3D genome organization, particularly at the regions it binds outside centromeres? Have you considered using approaches such as Hi-C to explore its role in local or global chromatin architecture?

A1: Absolutely yes to both questions. We think the evidence supports this possibility, and we would like to address this. But to do this really well, we think we need to first understand more about CENP-B binding to regions outside centromeres, which leads on perfectly to your next question.

Q2 : Do you think CENP-B engages in different interactions at centromeric versus non-centromeric regions? For instance, could differences in its interacting partners or post-translational modifications contribute to its distinct functions across these genomic contexts?

A2: Yes, we speculate that this is the case, and that there are other determinants – beyond hairpin-forming CCAAT box sequences – that regulate CENP-B binding and activity at chromosome arms. But unsurprisingly, the interactomes of CENP-B are dominated by centromere-associated proteins. Therefore, an approach that we are currently working on is the identification of separation-of-function mutations. This will allow us to explore the arm-specific functions of CENP-B without perturbing normal centromere activity, and potentially vice versa. Once we have these in hand, we can use them to look for binding partners, regulatory mechanisms, and impacts on chromatin architecture that are chromosome arm-specific.

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