Chapter 5. Nuclear actin-related proteins in epigenetic control

Abstract

The nuclear actin-related proteins (ARPs) share overall structure and low-level sequence homology with conventional actin. They are indispensable subunits of macromolecular machines that control chromatin remodeling and modification leading to dynamic changes in DNA structure, transcription, and DNA repair. Cellular, genetic, and biochemical studies suggest that the nuclear ARPs are essential to the epigenetic control of the cell cycle and cell proliferation in all eukaryotes, while in plants and animals they also exert epigenetic controls over most stages of multicellular development including organ initiation, the switch to reproductive development, and senescence and programmed cell death. A theme emerging from plants and animals is that in addition to their role in controlling the general compaction of DNA and gene silencing, isoforms of nuclear ARP-containing chromatin complexes have evolved to exert dynamic epigenetic control over gene expression and different phases of multicellular development. Herein, we explore this theme by examining nuclear ARP phylogeny, activities of ARP-containing chromatin remodeling complexes that lead to epigenetic control, expanding developmental roles assigned to several animal and plant ARP-containing complexes, the evidence that thousands of ARP complex isoforms may have evolved in concert with multicellular development, and ARPs in human disease.

Figure 5.1

Sectored colony color morphology of ARP4-defective yeast results from an epigenetic defect. (A) Sectored colony color phenotype resulting from the loss of ARP4 function in an ade2⁻ yeast strain containing the epigenetic reporter his4δ-ADE2 (Jiang and Stillman, 1996). Portions of two colonies where the his4δ-ADE2 reporter gene is “Off” (red) or “On” (white) are indicated with arrows. This reporter with a dysfunctional promoter is normally “Off” producing red colonies in ade2⁻ yeast cells. The reporter is turned “On” stochastically in arp4⁻ yeast cells. Photo, courtesy of David Stillman. (B) Normal HIS4 promoter and gene structure. (C) A model showing the structure of the his4δ-ADE2 epigenetic reporter gene in relation to possible changes in chromatin structure that render the gene transcriptionally “On” or “Off”.

Figure 5.2

Nuclear localization of five plant ARPs. The nuclear localization of Arabidopsis ARP4, ARP5, ARP6, ARP7, and ARP8 were demonstrated using ARP-class-specific monoclonal antibodies prepared against the various recombinant plant proteins or synthetic peptides. ARP4, ARP5, ARP6, and ARP7 are concentrated in the nucleoplasm, while ARP8 is concentrated in the nucleolus. For the ARP8 image, DNA staining with DAPI (red) is merged with the ARP8-specific monoclonal antibody immunostaining (green). A control strip of root cells with secondary antibody labeling is shown in the bottom panel.

Figure 5.3

Potential NLS, NES, and phosphorylation sites. (A) The proposed nuclear localization sequences (NLS) in various ARP4s are underlined and appear to be only moderately well conserved across the four eukaryotic kingdoms. The human (Hs), Arabidopsis (At), yeast (Sc), and protist (Tetrahymena thermophila, Tt) sequences are compared. (B) Proposed nuclear export sequences are compared among the yeast nuclear ARPs and actin and a consensus (CON) sequence is given. (C) Potential N-terminal phosphorylation sequences in ARP4 class members from the various kingdoms are compared to actin sequence. The conserved serine (S) and tyrosine (Y) residues that may be phosphorylated are underlined.

Figure 5.4

Phylogenetic relationships among nuclear ARPs from the four eukaryotic kingdoms. The phylogenetic relationships among the nuclear ARPs encoded by the animal (human, Hs), yeast (Saccharomyces cerevisiae, Sc), plant (Arabidopsis thaliana, At), and protist (Dictyostelium discoideum, Dd) genomes are illustrated. The four classes of nuclear ARPs—ARP4, ARP5, ARP6, and ARP8—that are generally conserved among animals, plants, fungi, and some protists are indicated. A few examples of orphaned ARPs are indicated by underlining. Clustal was used to align the sequences. The phylogram presented used the unweighted pair-group method with arithmetic means (UPGMA) to create the tree’s topography based on sequence similarity (Tamura et al., 2007). The neighbor joining tree building method also yields a tree with very similar, but not identical, branching patterns. Human β-actin and Arabidopsis ACTIN2 were used as conventional actin gene representatives from within the divergent families of animal and plant actins.

Figure 5.5

Coding sequence insertions in ARPs with respect to amino acid positions in conventional human β-actin. The locations of polypeptide insertions in the ARP4, ARP5, ARP6, and ARP8 classes that are conserved across all four eukaryotic kingdoms are shown. Numbered positions refer to amino acids in human β-actin. Insertions in yeast orphaned ARPs—ARP7 and ARP9—that are conserved in fungi are also shown for comparison.

Figure 5.6

Comparison of intron–exon structures for ARP4 genes across the four eukaryotic kingdoms. The intron/exon structure of ARP4 genes from Arabidopsis (At), human (Hs, Baf53a gene), protist (Dictyostelium discoideum, Dd), and yeast (Sc) genomes are compared. Coding exons are shown as white boxes, introns as lines, and transcribed but untranslated flanking regions (UTRs) in light gray boxes. The accession numbers of the particular transcript sequence compared are AT1G18450.1, BAG51043, XP_640964, and NP_012454, respectively, distinguishing these data from other possible transcript variants that exist for the plant and animal sequences. Conserved intron–exon junction positions are indicated by dashed lines. To generate these data, the intron positions identified from transcript and gene sequence alignments were compared to the amino acid sequence alignment of the four ARP4 protein sequences.

Figure 5.7

Nuclear ARP4 (Baf53) and actin bind the HSA domain of the Swi2-related Brg DNA-dependent ATPase in the mammalian Swi/Snf BRG chromatin remodeling complex. This model illustrates that β-actin and ARP4 bind Brg, and that the β-actin/ARP4/Brg subcomplex binds to a second subcomplex containing several other Brg proteins to form an active chromatin-remodeling machine (Lessard et al., 2007; Szerlong et al., 2008). A related model may be proposed for a large number of chromatin-active complexes. Two nuclear ARPs or a nuclear ARP and actin bind as heterodimers to the helicase-SANT (HSA) domain of the large Swi2-related DNA-dependent ATPase subunit in chromatin remodeling complexes or the Vid21-related helicase subunit in chromatin modifying complexes. Again, the ARP-containing subcomplex then binds a second subcomplex with a larger number of subunits.

Figure 5.8

ARP6- and ARP4-defective Arabidopsis plants misregulate the phase transition from vegetative to reproductive growth. ARP6-defective plants flower early when grown under long- or short-day growth conditions, while ARP4-defective plants flower early only when grown under long-day conditions. (A) Twenty-day-old WT and arp6-1 (null) and arp6-2 (strong knockdown allele) plants grown under long-day conditions. (B) Fifty-day-old WT and arp6-1 and arp6-2 plants grown under short-day conditions. (C) Twenty-day-old WT and an ARP4 RNA interference silenced line (ARP4-Ri) grown under long-day conditions. (D) Sixty-eight-day-old WT and ARP4-Ri plant grown under short-day conditions. Long day = 16 h light/24 h. Short day = 9h light/24 h. (E) A general outline depicting the flow of information for developmental pathways under ARP-dependent epigenetic control. (F) Specific model for the ARP4- and ARP6-dependent control of flowering time. ARP6-containing SWR1 complexes potentiate the expression of the central repressor of flowering FLC. In ARP6 mutants when FLC levels are down, the levels of the transcriptional activators of flowering FT, SOC1, LFY, and AP1 are up and the plants flower early. ARP4-mediated epigenetic control of other high-level regulators of flowering including PhyB and CO is proposed in this model.

Figure 5.9

ARP4- and ARP7-defficient Arabidopsis plants display delayed floral organ senescence and abscission. (A) WT retains sepals and petals on only four to five flowers when grown under long-day conditions. (B) A moderate ARP7 RNA interference knockdown line (7Ri) retains petals and sepals after fertilization and even after the fruits are fully developed. (C) A strong ARP7 RNAi line (right) is relatively sterile and retains floral organs on all flowers as compared to wild type (left). (D) Close-up examination of developing fruits in a moderate ARP4 RNA interference line (4Ri) reveals retention of sepals and petals for a longer period than wild type (WT, A). (E) A general pathway proposed for the ARP4- and ARP7-dependent epigenetic control of floral organ senescence. A large number of transcription factors are known to control floral senescence and it is likely that the influence of age, nutrients, and temperature are processed through ARP-dependent epigenetic mechanisms. ARP7-dependent chromatin remodeling activities function downstream of ethylene perception, whereas the sites of ARP4s activities are unclear (Meagher et al., 2007).

Figure 5.10

Nuclear ARP-defects alter the cell cycle and/or cell proliferation with effects on leaf morphology. ARP4-, ARP5-, ARP6-, and ARP7-deficient Arabidopsis plants all produce dwarf leaves, but the small organs result from different epidermal leaf cell morphologies. (A, D, E) ARP4-defective RNAi lines (4Ri) produce very small leaves composed of mixtures of small and tiny cells (E) compared to wild type (WT, D). (B, F) The ARP5-defective null mutant arp5-1 displays small, elongated leaves with mixtures of large and small poorly lobed cells and a significant excess of underdeveloped stomatal complexes relative to WT. (C, G) ARP7-defective RNAi lines (7Ri) develop small curled leaves composed of a few normal sized cells interspersed with large numbers of very small cells, as compared with the WT. (H–J) The ARP6 null mutant (arp6–1) produces small curled leaves composed of fewer cells, but of relatively normal cell sizes and with only slightly reduced number of lobes relative to wild type. (D–G, I, J) Scanning electron micrographs compare the epidermis of WT to ARP-deficient lines. All light and scanning microscope images were prepared using the largest rosette leaves from 3-week-old plants. (K) Possible pathways for epigenetic control of the cell cycle and endocycle are outlined. The phenotypes of ARP4-, ARP5-, ARP6-, and ARP7-defective plants suggest that cell division, cell expansion, and cell differentiation are under the influence of complex epigenetic control. By this model, ARP-containing chromatin remodeling machines respond to environmental influences and regulate the expression of transcription factors and signaling proteins controlling the cell cycle, cell proliferation, and cell and organ development.