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Actin-related protein 6: Biological Overview | References

Neurogenesis is initiated by basic helix-loop-helix proneural proteins. This study shows that Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression. Arp6 mutants exhibit reduced transcription in sensory organ precursors (SOPs) downstream of the proneural protein patterning event. This leads to retarded differentiation and division of SOPs and smaller sensory organs. These phenotypes are also observed in proneural gene hypomorphic mutants. Proneural protein expression is not reduced in Arp6 mutants. Enhanced proneural gene expression fails to rescue retarded differentiation in Arp6 mutants, suggesting that Arp6 acts downstream of or in parallel with proneural proteins. H2A.Z mutants display Arp6-like retardation in SOPs. Transcriptomic analyses demonstrate that loss of Arp6 and H2A.Z preferentially decreases expression of proneural protein-activated genes. H2A.Z enrichment in nucleosomes around the transcription start site before neurogenesis correlates highly with greater activation of proneural protein target genes by H2A.Z. It is proposed that upon proneural protein binding to E-box sites, H2A.Z incorporation around the transcription start site allows rapid and efficient activation of target genes, promoting rapid neural differentiation (Hsiao, 2023).

The basic helix-loop-helix (bHLH) proneural proteins are neural-inducing factors that confer neural fate to neuroepithelium or ectodermal cells. They promote neural fate determination, neural type specification and neural cell differentiation both in vivo and in vitro by transcriptional activation of a series of downstream genes. Nucleosome modification by histone acetyltransferase and demethylase and nucleosome relocation by the SWI/SNF remodeling complex are essential for regulation of proneural proteins to activate transcription in vertebrate cells (Hsiao, 2023).

Extensive studies in recent years have revealed that histone variant exchange alters the properties of the nucleosome core, thereby influencing chromatin-associated events, such as transcription, genome stability and DNA repair. The canonical H2A has two variants, H2A.Z and H2A.X, involved in nucleosome function and the DNA-damage response, respectively. H2A.Z typically constitutes 5-10% of the total H2A and is incorporated into a subset of nucleosomes at nonrandom locations throughout the genome. H2A.Z is enriched within a few nucleosomes surrounding the transcriptional start site (TSS), although the extent of incorporation varies between different genes (Hsiao, 2023).

One of the significant functions of H2A.Z is transcriptional activation. H2A.Z incorporation levels largely correlate with gene expression in the metazoa. It is also enriched in genes poised for activation in human cells. Mechanistically, H2A.Z is required to recruit RNA polymerase II (Pol II) to the promoter. The height of the +1 nucleosome barrier to Pol II inversely correlates with enrichment of H2A.Z. Besides transcriptional activation, H2A.Z is implicated in transcriptional repression, gene silencing, and heterochromatin formation and spreading (Hsiao, 2023).

H2A.Z is incorporated into nucleosomes by the highly conserved SWR1 complex. It partially unwraps DNA from the canonical histone core and catalyzes the replacement of an H2A-H2B dimer with an H2A.Z–H2B dimer. Actin-related protein 6 (Arp6) is a highly conserved component of the SWR1 complex. It physically interacts with the core ATPase of SWR1 and is essential for complex integrity and histone exchange activity. Arp6 is a member of the actin superfamily, and both Arp6 and nuclear actin are conserved members of the SWR1 complex in yeast and mammals (Hsiao, 2023).

Drosophila external sensory (ES) organ development is a classical model in which to study neurogenesis initiated by bHLH proneural proteins. Achaete (Ac) and Scute (Sc) were the first members of the highly conserved proneural protein family to be discovered. Both proteins are first expressed in proneural stripes to endow neural potential to the cells. Subsequently, high-level Ac and Sc are restricted to single sensory organ precursors (SOPs) to promote precursor determination, selection and differentiation by transcriptional activation of an array of downstream genes. This paper reports the crucial role of Arp6-mediated H2A.Z replacement for rapid SOP differentiation and division downstream of the proneural protein patterning event. Arp6 is required for efficient transcription of Ac- and Sc-target genes in SOPs. Genetic analyses suggest that Arp6 functions downstream of or in parallel with proneural proteins to activate the SOP differentiation program. Transcriptomic analyses showed that both Arp6 and H2A.Z are preferentially required for proneural protein-induced transcription. Loss of H2A.Z did not reduce proneural protein binding to high-affinity E-box sites, suggesting that E-box binding is independent of H2A.Z enhancer incorporation. phyl is a proneural protein target gene that requires Arp6 and H2A.Z for its maximal expression. H2A.Z was incorporated into the nucleosome around the TSS of

phyl in the absence of proneural proteins, and recruitment of proneural proteins to the E-box enhances H2A.Z incorporation through Arp6. Database analysis of the H2A.Z replacement at the +1 nucleosome preceding neurogenesis revealed a strong correlation between H2A.Z enrichment and efficient gene expression by proneural proteins. It is proposed that, in the absence of H2A.Z replacement at the +1 nucleosome, proneural proteins still bind to the cognate target sites with high affinity but initiate transcription at a slower rate, thus retarding precursor differentiation (Hsiao, 2023).

bHLH proneural proteins act as pioneer factors to recognize cognate E-box sequence and activate target gene expression for neural determination and differentiation. The current data demonstrate that the histone variant H2A.Z is a crucial chromatin factor for proneural protein-induced maximal gene expression: (1) loss of Arp6 and H2A.Z led to prolonged SOP development and smaller ES organs, the phenotypes also observed in ac sc hypomorphic mutants; (2) SOPs and ES organs were almost eliminated in Arp6 and ac sc hypomorphic double mutants; (3) transcription of proneural protein target genes phyl and sca in SOPs were markedly reduced in Arp6 mutants; and (4) transcriptomic analysis revealed that loss of Arp6 and H2A.Z preferentially diminished gene expression induced by Ac-Da compared with the constitutively activated genes. Unlike the histone acetyltransferase CBP and the chromatin remodeler Brg1 in Xenopus and mouse, which are required for the generation of neural progenitors and neurons by proneural proteins, this study found that Arp6-mediated H2A.Z incorporation specifically influences precursor differentiation rate without affecting fate determination and formation of SOPs. Therefore, histone variant H2A.Z exchange plays a distinct role in the regulation of proneural protein-induced neurogenesis compared with histone modification and nucleosome remodeling (Hsiao, 2023).

This analyses suggest that Arp6-mediated H2A.Z replacement at the +1 nucleosome facilitates proneural protein target gene expression by both proneural protein-dependent and -independent mechanisms. From the S2 cell studies, proneural proteins were found to enhance H2A.Z incorporation on its target gene phyl and loss of Arp6 diminished the elevated H2A.Z replacement. These results, together with the physical interaction between Arp6 and proneural proteins, suggest that proneural proteins act upstream and can recruit Arp6 to enhance H2A.Z incorporation. H2A.Z enrichment at the +1 nucleosome before neurogenesis was found to be highly correlated with the activation of proneural protein target genes by H2A.Z and Arp6 is required for this basal H2A.Z incorporation, at least to >phyl. In addition, Arp6 and H2A.Z do not significantly influence E-box binding. These results suggest a parallel action between Arp6 and proneural proteins in the activation of proneural protein target genes. Genetic analyses showed that Ac overexpression failed to rescue timely SOP differentiation in Arp6 mutant clones, indicating that the absence of Arp6 could not be replaced by enhanced Ac expression. The genetic and biochemical data support the suggestion that Arp6 acts both downstream and in parallel with proneural proteins to facilitate proneural protein-induced gene activation (Hsiao, 2023).

Studies in the past two decades have demonstrated that nuclear actin and nuclear ARPs are crucial components of chromatin remodeling and modifying complexes. The nuclear actin-Arp4 dimer participates in chromatin complexes, such as SWR1, Ino80 and BAF. This study has shown that nuclear actin is important for proneural protein-mediated gene activation in SOPs and S2 cells; knockdown of both Actin 5C and Actin 42A disrupted SOP formation and overexpression of nuclear β-actin increased Ac/Da-dependent transcription in S2 cells. Given that nuclear actin, Arp4 and Arp6 are the core components of SWR1, the current results suggest that one of the major functions of nuclear actin family proteins in neurogenesis involves H2A.Z exchange. A recent study also reveals that the β-actin level is particularly important for enhancing chromatin accessibility at the promoter region of genes involved in the regulation of neuron differentiation in mouse cells. Thus, analyses of nuclear actin and ARPs in SOPs suggest a highly conserved mechanism of the nuclear actin family protein in the modulation of gene expression in neural development (Hsiao, 2023).

Proneural proteins are master regulators of ES organ development. Their spatial and temporal expression patterns determine where and when SOP fate determination and differentiation occur. In developing nota, Ac and Sc are expressed in longitudinal proneural stripes at 6-9 h APF in response to patterning cues from Delta-Notch signaling. The current results showed that proneural patterning and ac sc expression in cultural cells was largely unaffected by loss of Arp6-mediated H2A.Z incorporation. At later stages, proneural protein is crucial for precursor selection from proneural cells; single SOPs are selected and arranged in regular arrays by several proneural protein-mediated mechanisms: (1) the proneural target gene phyl is expressed at a high level in SOPs to facilitate lateral inhibition by Notch signaling; (2) proneural protein target gene sca is expressed in proneural clusters and SOPs to mediate long-range precursor spacing; and (3) Notch target genes E(spl)m7 and m8 were activated by proneural proteins in proneural stripes to mediate SOP selection and spacing. Following SOP selection, proneural proteins in single SOPs are gradually downregulated via negative feedback regulation by phyl to promote G2-M progression. SOPs also require proneural protein target gene sens for timely SOP division. Therefore, insufficient target gene expressions, caused either by reduced H2A.Z replacement in Arp6 and H2A.Z mutants or by low ac sc expression in acsbm mutants, lead to retarded SOP formation and division, and, ultimately, smaller ES organs (Hsiao, 2023).

These results showed thatsbmpxhy phyl and sens are activated by Arp6 in SOPs. Although phyl and sens are also required for binary cell fate determination of SOP daughter cells, cell fate transformation was almost not observed in Arp6 and H2A.Z mutants. There are two possible explanations. First, phyl and sens were expressed in lower but sufficient levels in SOP daughters to sustain normal cell fate determination in Arp6 and H2A.Z mutants. Second, Arp6 and H2A.Z do not modulate phyl and sens expression in SOP daughter cells. Once Phyl and Sens reached sufficient levels in mature SOPs in Arp6 mutants, their levels in SOP daughter cells (pIIa and pIIb cells) were not reduced in mutant clones. Combined with the transcriptomic result that H2A.Z is largely dispensable for the expression of non-Ac-Da-inducible genes, it is hypothesized that Arp6-mediated H2A.Z incorporation has little to no effect on phyl and sens expression in SOP daughter cells (Hsiao, 2023).

The transcriptomic analysis revealed that H2A.Z is preferentially required for Ac-Da-induced gene activation. By contrast, recent studies also showed that H2A.Z is essential for the expression of constitutively activated genes but not for transcriptional activator Zelda-dependent transcription during the zygotic genome activation. In addition, H2A.Z plays diverse roles in regulating DNA binding by transcriptional factors. H2A.Z could have positive, negative , or no effect (this study) on the DNA binding. (Hsiao, 2023).

Analyses of H2A.Z incorporation among Ac-induced genes support the hypothesis that differential H2A.Z replacement at the +1 nucleosome contributes to proneural target gene activation by H2A.Z. Several mechanisms have been proposed to mediate differential incorporation by SWR1. The yeast SWR1 complex preferentially binds promoters containing a more extended nucleosome-free region located between the +1 and −1 nucleosomes. DNA methylation prevents H2A.Z incorporation in Arabidopsis. Histone H3 acetylation contributes to preferential SWR1 recruitment from yeast to human. Therefore, intrinsic chromatin architecture and modification might further determine the responsiveness of proneural target genes to H2A.Z-dependent transcriptional activation (Hsiao, 2023).

Although Arp6 is essential for maintenance of H2A.Z levels in developing cells, as shown by the elimination of H2A.Z in a strong loss-of-function Arp6¹ clone, H2A.Z incorporation was only partially compromised in S2 cells by the knockdown of Arp6 and YL-1, another core component of the SWR1 complex. It is hypothesized that RNAi-mediated silencing of Arp6 and YL-1 is not efficient enough to abolish most SWR1 activity in S2 cells, leading to incomplete elimination of H2A.Z from nucleosomes in knockdown cells (Hsiao, 2023).

In mammals and yeast, the catalytic subunits of the SWR1 complex and NuA4 complex are encoded by distinct genes. In Drosophila, recent studies have revealed that the core ATPase of SWR1 complex and NuA4 complex are encoded by different isoforms of dom, thus complicating the previous molecular interpretation of dom mutant phenotypes. Analysis of the SWR1 complex component has revealed that Arp6 is the sole SWR1-specific component from yeast to human. Thus, Arp6 mutants are an excellent tool with which to verify and further investigate SWR1-specific function in Drosophila (Hsiao, 2023).

Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin

Actin-related proteins (Arps), which share a basal structure with actin isoforms but possess different functions, have been identified in a wide variety of organisms. The Arps are classified into subfamilies based on the relatedness of their sequences and functions. Recently, several Arp subfamilies have been shown to be localized in the nucleus and included in protein complexes involved in the organization of chromatin structure, for example, in chromatin remodeling and histone acetyltransferase complexes. A member of the Arp6 subfamily in Drosophila, dArp6, is localized on centric heterochromatin together with heterochromatin protein 1 (HP1). This study has identified the first examples of the Arp6 subfamily in vertebrates, novel human and chicken Arps, hArp6 and gArp6, respectively. They are closely related to each other (98% similar) and show apparent similarity to dArp6 (70%). In addition, the hArp6 gene possesses evolutionarily conserved exon/intron structures compared with genes for members of the Arp6 subfamily in invertebrates. Like Drosophila dArp6, gArp6 is expressed abundantly in the early developmental stages, when heterochromatin condensation and nuclear maturation occur. The finding of a conserved Arp6 subfamily in vertebrates will contribute to the understanding of molecular mechanisms of heterochromatin organization (Kato, 2001).

Identification of a divergent actin-related protein in Drosophila

The actin-related proteins (ARPs) have primary sequence homology to actin, have no homology to other proteins and, unlike the conventional actins, are clearly divergent. An ARP has been identified in Drosophila that has approximately 30% amino acid identity to most actins, making it the most divergent yet reported. It is also quite divergent from all other ARP sequences. When the Drosophila ARP is aligned with actin it contains sequence insertions, as is the case with all other ARPs. The unique location of the insertions, as well as its overall divergence, indicates it may represent a new isotype. Only one gene was detected by hybridization to both genomic DNA and polytene chromosomes; the location of the gene is 13E on the X chromosome. A transcript of 1350 bases was detected at all stages of development. This transcript was relatively abundant during early embryogenesis, decreasing during the later stages of embryogenesis and increasing again in larvae and adults (Frankel, 1994).

Functions of Arp6 orthologs in other species

Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1

Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. The predominant nuclear localization of another Arp subfamily, Arp6, occurs in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. These data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin (Ohfuchi, 2006).

Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6

The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans. A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin. Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog. However, the function of S.pombe Arp6 has not been characterized yet. Deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing. Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region. However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei. The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+). Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+). These results suggest that Arp6 and Swi6 function independently at telomere ends. The Arp6-mediated repression mechanism is proposed to work side by side with Swi6-based telomere silencing in S.pombe (Ueno, 2004).

MPK4-mediated phosphorylation of PHYTOCHROME INTERACTING FACTOR4 controls thermosensing by regulating histone variant H2A.Z deposition

Plants can perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis is well established, although the complete regulatory pathway involved in thermosensing remains elusive. This study established that an increase in temperature from 22 to 28 degrees C induces upregulation and activation of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) in Arabidopsis (Arabidopsis thaliana), subsequently leading to the phosphorylation of PIF4. Phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6), which is required for mediating the deposition of histone variant H2A.Z at its target loci. Furthermore, variations in ARP6 expression were identified in PIF4 phosphor-null and phosphor-mimetic seedlings affect hypocotyl growth at 22 and 28 degrees C by modulating the regulation of ARP6-mediated H2A.Z deposition at the loci of genes involved in elongating hypocotyl cells. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in a temperature-dependent manner. Taken together, these findings highlight the regulatory mechanism of thermosensing by which MPK4-mediated phosphorylation of PIF4 affects ARP6-mediated H2A.Z deposition at the genes involved in hypocotyl cell elongation (Verma, 2024).

Search PubMed for articles about Arp6 Drosophila

Frankel, S., Heintzelman, M. B., Artavanis-Tsakonas, S., Mooseker, M. S. (1994). Identification of a divergent actin-related protein in Drosophila. J Mol Biol, 235(4):1351-1356 PubMed ID: 8308899

Hsiao, Y. L., Chen, H. W., Chen, K. H., Tan, B. C., Chen, C. H. and Pi, H. (2023). Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation. Development 150(5). PubMed ID: 36897355

Kato, M., Sasaki, M., Mizuno, S., Harata, M. (2001). Novel actin-related proteins in vertebrates: similarities of structure and expression pattern to Arp6 localized on Drosophila heterochromatin. Gene, 268(1-2):133-140 PubMed ID: 11368909

Ohfuchi, E., Kato, M., Sasaki, M., Sugimoto, K., Oma, Y., Harata, M. (2006). Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1. Eur J Cell Biol, 85(5):411-421 PubMed ID: 16487625

Ueno, M., Murase, T., Kibe, T., Ohashi, N., Tomita, K., Murakami, Y., Uritani, M., Ushimaru, T., Harata, M. (2004). Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6. Nucleic Acids Res, 32(2):736-741 PubMed ID: 14757838

Verma, N., Singh, D., Mittal, L., Banerjee, G., Noryang, S., Sinha, A. K. (2024). MPK4-mediated phosphorylation of PHYTOCHROME INTERACTING FACTOR4 controls thermosensing by regulating histone variant H2A.Z deposition. Plant Cell, 36(10):4535-4556 PubMed ID: 39102893 Biological Overview

date revised: 26 March 2025

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