Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

Abstract

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.

Figure 1. MSL3-GFP targets to the periphery of chromosomes in live embryonic cultured cells expressing MSL3-GFP and His2AvDmRFP1 at different stages of the cell cycle.

For all pseudo-colored images: RFP1, cyan; GFP, magenta. (A–F) Rows from top to bottom: interphase, prophase, prometaphase, metaphase, anaphase (side view), and anaphase (cross-section view). Columns from left to right: His2AvDmRFP1-labeled chromatin, MSL3-GFP, superimposed mRFP1 (cyan) and GFP (magenta) signals, 3-fold higher magnification of the labeled X chromosome arm. All images were deconvolved. Bars: 1 µm – (F, third column), G, H, I; 0.5 µm – (F, last column).

Figure 2. Active genes localize to the periphery of mitotic chromosomes in mitosis starting late prophase in fixed cells of embryonic cultures.

Columns: first – total DNA labeled by His2AvDmRFP1; second – MSL3-GFP signal; third – superimposed, pseudo-colored GFP, and RFP channels; right – 3-fold higher magnification of the labeled region from the third column. In embryonic cultured cells expressing MSL3-GFP and His2AvDmRFP1 and fixed in early prophase (A), GFP and mRFP1 signals do not overlap. Rather, they complement each other (shown with arrowheads) within the partially condensed prophase chromosome arm, an extended structure 3–5 µm long and ∼200 nm wide. At early prophase, MSL3-GFP signal is often found to run across the entire width of condensing chromosomes. In addition, domains of MSL3-GFP signal are sandwiched between the domains of His2AvDmRFP1 signal without overlapping. In contrast, late prophase (B) and metaphase (C) distributions of MSL3-GFP are peripheral and locate to the periphery of the condensed chromosomal arms in all observed cells. Bars: 1 µm – first three columns and 500 nm – right column.

Figure 3. MSL3-GFP, peripheral during anaphase, remains mostly peripheral during decondensation.

(A, B) MSL3-GFP does not redistribute during post-mitotic decondensation and interphase. The numbers represent minutes after beginning of observation. The first images of (A) and (B) are captured in mid-anaphase: peripheral MSL3-GFP is shown. (A) Anaphase to interphase dynamics of MSL3-GFP labeled X chromosomes in live 3^rd instar larval brains. (B) Anaphase to interphase dynamics of MSL3-GFP labeled X chromosomes in live embryonic cultures. Arrowheads point to apparent halves of MSL3-GFP signal, with an area inside devoid of MSL3-GFP. In many cells of live embryonic cultures (C) and tissues (D), MSL3-GFP labeled chromatin loci in interphase NBs form a typical pattern: a rim of MSL3-GFP signal around a region with no or little GFP signal. Gray-scale images in (C) and (D) are 3-fold magnifications of center colored panels. Numbers represent seconds (C) and minutes (D) after beginning of observation. All images, except for (B), were deconvolved. Bars: 1 µm.

Figure 4. The density of specific histone modifications varies across metaphase and anaphase chromosomes.

(A–C) Double antibody staining against specific histone modifications in fixed embryonic cultures is radially non-uniform in mitotic chromosomes. In (A) and (B), examples are shown of cells stained with DAPI for DNA (left images), anti-H3K27me1 (center images), and anti-H3K4me2,3 (right images) antibodies, (C) DAPI (left), anti-H3K4me2,3 (middle), and anti-H4K20me1 (right) antibodies. (A) An optical cross-section of anaphase chromosomes oriented along the optical axis; (B) side view of metaphase chromosomes; (C) side view of anaphase chromosomes. Arrowheads point to regions magnified 2.5-fold in insets. Insets: (A,B) DAPI (cyan) and anti-H3K27me1 (magenta) – left column; anti-H3K27me1 (cyan) and anti-H3K4me2,3 (magenta) – middle column; DAPI (cyan) and anti-H3K4me2,3 (magenta) – right column. (C) DAPI (cyan) and anti-H3K4me2,3 (magenta) – left column; anti-H4K20me1 (cyan) and anti-H3K4me2,3 (magenta) – middle column; DAPI (cyan) and anti-H4K20me1 (magenta) – right column. (D) Profiles for antibody stained chromosomes along lines marked by yellow segments in (A–C) in the same order. In interphase chromosomes, anti-MSL2 and anti-H3K4me2,3 signals marking active loci occupy regions spatially distinct from anti-H3K27me1 and anti-H4K20me1, demonstrating that tightly compacted mitotic chromatin is accessible to antibodies. Images were deconvolved. Bars: 1 µm – (C, D).

Figure 5. In fixed, antibody stained interphase cells from embryonic cultures anti-MSL2 (A) and anti-H3K4me2,3 (B) signals localize to the periphery of chromatin domains as detected by DAPI staining, while anti-H3K27me1 (C) and anti-H4K20me1 (D) signals largely overlap with chromatin.

Columns from left to right: DAPI for DNA, antibody staining, pseudo-colored superimposition of DAPI (cyan) and immunofluorescence (magenta) signals, 3-fold expanded region of the superimposition. Bars: 1 µm – (D) (third column); 0.5 µm – (D) (right column).

Figure 6. Formation of a radially non-uniform anaphase chromosome.

Large-scale re-arrangements inside 100–200 nm wide early prophase fibers (in which MSL3-GFP signals are often found throughout the entire chromosome width) result in peripheral localization of active sequences in ∼500 nm wide metaphase and anaphase chromatids.