Histone H4 represses histone gene expression during the cell cycle

If all the DNA in a human cell was stretched out end to end, it would be roughly six feet long. That’s a lot of genetic information to pack into a cell that is, on average, one-fifth the size of a grain of salt. Cells achieve this by tightly packaging DNA around groups of proteins called histones. Each group contains two sets of the four core histones: H2A, H2B, H3, and H4. Together, the DNA and histone proteins form nucleosomes, which assemble into chromatin. Beyond packaging DNA, histones protect DNA from damage, control which genes are expressed by a cell, and certain variants can even orchestrate DNA damage repair. During cell division, the genome must be duplicated and repackaged with histone proteins to be correctly split between the two new cells. This process depends on maintaining correct ratios of newly synthesized histone proteins to newly replicated DNA.

Histone protein levels are controlled by precise transcription of histone genes by RNA polymerase II (RNAPII). During DNA replication, this transcription can be fine-tuned by activating or repressive chromatin marks. Despite knowing the location of these genes and some of the molecular players involved in their transcription, how histone gene expression is regulated during DNA replication is a persistent question for scientists. To address this question, researchers in the Henikoff Lab, led by Dr. Kami Ahmad, used chromatin profiling and genetically engineered flies to characterize potential regulators of this process.

In Drosophila, or fruit flies, there are 100 tandemly repeated histone-encoding genes located in a specific region of the nucleus called the histone locus body. These histone genes are subjected to random silencing because of the numerous repeats, and the high copy number of each gene makes genetic knockout of any particular histone extremely difficult. To overcome this problem, the team used 12XWT flies, where the entire native histone locus body has been deleted and replaced with a construct carrying 12 copies of the histone repeat units. In 12XWT flies, all histone genes should be active, making it easier to compare the repressed gene expression observed in wild type flies to active gene expression.

The team next compared RNAPII occupation at each histone gene between wild type and 12XWT flies. They found significantly more RNAPII at each histone gene in 12XWT flies compared to wild type flies, indicating that the histone genes in 12XWT flies are more heavily transcribed. Consistent with this finding, there were more activating chromatin marks on the histone genes in the 12XWT flies, whereas repressive chromatin marks were enriched on histone genes in wild type cells. To measure histone protein expression, researchers expressed fluorescent His2ADendra2 and His3Dendra2 fusion proteins in both fly models. They found that fluorescence of both proteins was significantly higher in the 12XWT flies, indicating that histone expression is partially repressed in wild type flies.

To tease the mechanism underlying histone gene repression apart, Ahmad silenced several different chromatin-modifying genes in wild type flies expressing the His2ADendra2 or His3Dendra2 proteins to see if the silenced genes could de-repress histone expression and increase Dendra2 fluorescence. Surprisingly, none of these silenced genes impacted protein fluorescence, indicating that they were likely not involved in histone gene repression. The group next performed the same experiment with the histone genes themselves. They found that selectively silencing H4 increased His2ADendra2 and His3Dendra2 fluorescence, suggesting that cells measure demand for all histones based on H4 expression during DNA replication.

H4 canonically exists in a complex with the other histone proteins, so the team was initially surprised that silencing only H4 impacted overall histone gene expression. However, other groups have reported monomeric histone proteins in Drosophila and human cells, so they tested whether H4 could localize to the histone locus body on its own. They found that H4 localized as a small dot in the nuclei of cells during DNA replication but not during other parts of the cell cycle. Even though H4 is found throughout the chromatin, the team did not observe widespread H4 staining, indicating that their stain recognizes a monomeric form of H4. Other experiments showed that H4 abundance increases where RNAPII abundance decreases. These data support the idea that monomeric H4 has a role in repressing all histone genes.

Unlike Drosophila, human histone genes are distributed across four histone locus bodies throughout the genome. Still, histones are ancient proteins, and any mechanisms governing their expression in Drosophila should be conserved in humans. To test this, Ahmad used a human cell line to map H4 localization during cell division. He found that, just like in flies, H4 localizes to the histone locus bodies of human cells, confirming that H4-mediated control of histone gene expression is evolutionarily conserved.

Histones and their genetic regulation are indispensable to the most basic biological processes that sustain life. How, then, have the mechanistic details of histone regulation gone undiscovered for all these years? Initially, the team started this work trying to learn more about gene silencing during development. “Once we started getting results, we realized we needed to look back at our textbooks. We never actually answered this question!” says Ahmad. Through this work, the team has put forth a model where chromatin packaging during DNA replication consumes soluble histones, and once this process ceases, soluble H4 accumulates to repress histone gene expression. “One of the most valuable things about the paper is bringing these questions back up…I think this is going to be just one example of how [DNA replication and histone gene expression] are linked,” continues Ahmad. With the question fresh in researchers’ minds, only time will tell what new molecular details are unveiled about these ancient processes.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Dr. Steven Henikoff contributed to this research.

This work was supported by funding from the Howard Hughes Medical Institute.

Ahmad K, Wooten M, Takushi BN, Vidaurre V, Chen X, Henikoff S. 2025. Cell-cycle-dependent repression of histone gene transcription by histone H4. Nat Strut Mol Biol. 33(1):145–156. https://doi.org/10.1038/s41594-025-01731-1.