Early Diverging Fungus Mucor circinelloides Lacks Centromeric Histone CENP-A and Displays a Mosaic of Point and Regional Centromeres

Abstract

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Keywords: CENP-A; Grem-LINE1; Mis12 complex; Mucoromycotina; RNAi; centromere; early-diverging fungi; kinetochore; mosaic; retrotransposon.

Figure 1.. Distribution of the kinetochore complex across the Mucoromycotina subphylum.

Concise kinetochore schematic showing the most conserved protein in eukaryotes is shown on the left corresponding to the kinetochore proteins analyzed in matrix on the right. The matrix displays the presence or absence of 26 kinetochore across a cladogram of 51 fungal lineages (top) to show the relationships among them. fungal phylum is represented and color-coded, differentiating the Mucoromycota into Glomeromycotina, Mortierellomycotina, and Mucoromycotina subphyla to provide a emphasis on the latter. Arrows at divergence events in the cladogram mark the hypothetical loss of proteins CENP-A (red) and CENP-C (blue) in these clades. See also Figure S1, Tables S1 and S4.

Figure 2.. Inner and outer kinetochore proteins colocalize in M. circinelloides nuclei in the absence of CENP-A and CENP-C.

(A) Graphical representation of Mis12, Dsn1, and CENP-T sequence features showing individual Pfam domains PF05859, PF08202, PF15511 respectively identified in M. circinelloides. (B) Schematic to represent C-terminal tagging of histone H3 and kinetochore proteins with mCherry (red circles), eGFP (green circles). (C-F) Confocal microscope imaging showing the cellular localization of well-conserved mCherry-tagged outer kinetochore proteins Mis12 (C, E) and Dsn1 (D, F), eGFP-tagged histone H3 (C, D), and eGFP-tagged inner kinetochore CENP-T (E, F) in pre-germinated spores of M. circinelloides strains expressing fluorescent fusion proteins. (G) Time-lapsed confocal image displaying the cellular localization of mCherry-tagged Mis12 and eGFP-tagged histone H3 in a germinative tube sprouting from a spore. A discontinuous white perimeter outlines the germinative tube in the fluorescence images, and a white square indicates a nuclear division event. Cartoon for the zoomed image represents the localization and signal intensity of Mis12-mCherry in the nucleus. A calibrated scale (white bar) is provided for size comparison (5 μm) and mCherry fluorescent signal is colored as magenta in the merged images in C-G. See also Figures S2 and S3, Table S2, and Video S1.

Figure 3.. M. circinelloides displays nine centromeres.

(A) Putative kinetochore-binding regions showing color-coded enrichment of immunoprecipitated DNA (IP DNA) from Mis12 and Dsn1 mCherry-tagged strains compared to their corresponding input (Input DNA) and binding controls (Beads only). 1 kb flanking sequences from the center of the enrichment peak are shown. Major (M) and minor (m) kinetochore-binding regions are indicated. Each kinetochore-protein data was obtained from a pool of duplicated IP DNA samples. (B) Scaled ideogram of the nine scaffolds (white) of M. circinelloides genome assembly that contain a kinetochore-binding region (blue), showing the telomeric repeats (red). See also Figure S4 and Data S1.

Figure 4.. The centromeres of M. circinelloides are short, AT rich and harbor a conserved DNA motif.

(A) Size distribution of the nine core centromere (CC) regions. (B) GC content across the nine core centromere (CC) sequences. The median (red line) and standard deviation (black lines) are shown in A and B. (C) 41 bp centromere-specific DNA motif conserved in all nine CC regions and absent in the remainder of the genome. A score of 2 bits in the logo indicates that the nucleotide is present at that position in all nine CC regions. (D) A genomic view of CEN11 illustrating all nine core centromere regions. Kinetochore-binding region enrichment (IP coverage) as the average of both immunoprecipitation signals minus Input and beads only controls (left axis, blue), GC content across the region (right axis, black), and the centromere-specific DNA motif described in e (red) are shown. See also Figure S4 and Data S1.

Figure 5.. M. circinelloides core centromeres are present in large ORF-free pericentric regions having retrotransposable elements regulated by the Dicer-dependent RNAi pathway and bind H3 nucleosomes.

(A) Genomic sequences lacking annotated genes (light blue) that flank the CC regions (dark blue) served as the reference points to calculate the length both upstream and downstream. Black circles at either end of the regions indicate an abrupt, non-telomeric end of scaffold. (B) Schematic of the Grem-LINE1 interspersed in the pericentromeric regions. Open Reading Frames (ORF) and protein domains predicted from their coding sequences are shown as colored boxes [Zf, zinc finger (PF00098 and PF16588); AP, AP endonuclease (PTHR22748); RVT, reverse transcriptase (PF00078); and zf-RVT, zinc-binding in reverse transcriptase (PF13966)]. (C) CEN4 is exemplified by low GC content, lack of genes and transcripts, which are shown in different tracks displaying the kinetochore-binding region enrichment (IP, an average of both IP DNA signals minus Input and beads only controls), annotation of genes (red blocks) and transposable elements (light blue blocks), CEN-specific DNA motif position (vertical line) and direction (arrow), GC content, and transcriptomic data of mRNA (green) and sRNAs (red) in M. circinelloides wild-type, ago1, and double dcl1 dcl2 deletion mutant strains after 48 h of growth in rich media. (D) Genomic location of CEN2 showing the regions studied for histone H3 occupancy as labelled and colored rectangles. ChIP assays were performed using polyclonal antibodies against histone H3. Primers were designed for CC2 region, pericentric regions (1L, 2L, 1R, 2R), flanking ORFs (ORF-L, ORF-R) and a far-CEN control ORF ~2 Mb away from CEN2 (Far-CEN). IP samples were analyzed by real-time PCR using these primers. The y-axis denotes the qPCR value as a percentage of the total chromatin input with standard error mean (SEM), from each region tested. The experiment was repeated three times with similar results. See also Figures S5 and S6, Tables S2 and S3, and Data S1.

Figure 6.. M. circinelloides possesses a mosaic of point and regional centromeres.

Length plot of the core centromere (kinetochore-bound region, blue circles) and the full centromere (defined as the region required for centromere function, red dots) of representative fungi belonging to the Mucoromycota (green panel), Ascomycota (blue panel) and Basidiomycota (purple panel). The size axis is divided in short (≤1 kb, gray area) and long regions (>1 kb, brown area). The centromeres of M. circinelloides (CEN5), S. cerevisiae (CEN3 [7]), Candida albicans (CEN7 [39]), C. tropicalis (Scnt 3 [40]), Magnaporthe oryzae Guy11 (CEN2 [49]), S. pombe (CEN2 [52]), and C. neoformans (CEN10 [23]) are used for comparison and classified as point, regional and mosaic centromeres according to their structural features. In organisms where functionality of the centromere regions could not be experimentally determined, the ORF-free region spanning the kinetochore binding region is considered as the full centromere. Line diagrams represent the structural features of each centromere (drawn to scale). The ORF-free region at the centromere is defined by black vertical boundaries and flanking ORFs are shown as red arrows. Core centromeres (rectangles); kinetochore-bound regions (blue area); centromere-specific DNA motifs next to an AT-rich core (green lines); Grem-LINE1, Grasshopper, GYMAG, Maggy, Tcn retrotransposons (black arrows); inverted repeats (purple arrows); and other DNA repeats (light and dark orange arrows) are shown. Repeats/retrotransposons sizes are not drawn to scale. CC, core centromere; CDE, centromere DNA elements; LR, left repeat; RR, right repeat; otr, outer repeats containing dg/dh elements; imr, innermost repeats; cnt, central core.