Bookmarking by specific and nonspecific binding of FoxA1 pioneer factor to mitotic chromosomes

Abstract

While most transcription factors exit the chromatin during mitosis and the genome becomes silent, a subset of factors remains and "bookmarks" genes for rapid reactivation as cells progress through the cell cycle. However, it is unknown whether such bookmarking factors bind to chromatin similarly in mitosis and how different binding capacities among them relate to function. We compared a diverse set of transcription factors involved in liver differentiation and found markedly different extents of mitotic chromosome binding. Among them, the pioneer factor FoxA1 exhibits the greatest extent of mitotic chromosome binding. Genomically, ~15% of the FoxA1 interphase target sites are bound in mitosis, including at genes that are important for liver differentiation. Biophysical, genome mapping, and mutagenesis studies of FoxA1 reveals two different modes of binding to mitotic chromatin. Specific binding in mitosis occurs at sites that continue to be bound from interphase. Nonspecific binding in mitosis occurs across the chromosome due to the intrinsic chromatin affinity of FoxA1. Both specific and nonspecific binding contribute to timely reactivation of target genes post-mitosis. These studies reveal an unexpected diversity in the mechanisms by which transcription factors help retain cell identity during mitosis.

Figure 1.

Diverse modes of hepatic transcription factor binding to mitotic chromosomes. (A,B) GFP fluorescence in live HUH7 hepatoma cells visualized by deconvolution microscopy with or without nocodazole for mitotic arrest. (A) GFP and GFP/bright-field (BF) views showing that GFP-FoxA1, GFP-FoxA1 DBD, and GFP-H1o remain quantitatively bound to mitotic chromosomes. (B) A portion of the cellular GATA4 and HMGB1 is released from mitotic chromosomes in nocodazole-treated cells; C/EBPα becomes unstable, yet a portion remains bound; and c-Myc and NF1 are excluded from the mitotic chromosomes.

Figure 2.

FoxA1 in mitosis occupies the most strongly expressed and strongly bound genes in asynchronous HUH7 cells. (A) Peaks were pooled from three replicate ChIP-seq samples, revealing 3509 asynchronous FoxA1-binding sites, of which 546 are also bound in mitosis. (B) FoxA1 ChIP-seq data tracks and ChIP-qPCR confirmation in independent chromatin samples with signals normalized to input and per million sequence reads. Red arrowheads depict sites of mitotic binding, and those that were verified by qPCR are shown on the right. The circle indicates the negative control site. The break in the HNF4a and TTR bar graphs is to accommodate different scales for the asynchronous (as.) and mitotic (mi.) data. (C) FoxA1 ChIP-seq signals over all sites bound in mitotic and asynchronous cells compared with all sites bound only in asynchronous cells; the signal is normalized to input DNA quantity and the total number of aligned sequence reads. FoxA1 binding is much stronger to sites in mitotic and asynchronous cells than to sites bound in asynchronous cells only. (D) Common FoxA-binding motif at sites bound in mitosis versus asynchronous only. (E) Nearly all of the FoxA1-bound sites in mitosis are associated with genes either within the transcribed region or <20 kb upstream. (F) Box and whisker plots showing that genes bound by FoxA1 in mitotic and asynchronous cells correspond to those more highly expressed genes in hepatoma cells; (***) P < 10⁻¹⁵. (G) FoxA1 remains bound to hepatic transcription factor genes in mitosis.

Figure 3.

FoxA1-bound sites in mitotic cells have higher predicted nucleosome occupancy compared with sites bound only in asynchronous cells. Average INOS for FoxA1 3509 ChIP-seq peaks seen only in asynchronous HuH7 cells and 544 peaks seen in mitotic cells within ±750 bp from the center of the peak; also shown are 100,000 sequences selected at random from human genome (hg18). The average INOS profiling of FoxA1 in mitotic HuH7 cells is higher than seen in asynchronous cells (t-test, P = 9.5 × 10⁻⁶).

Figure 4.

Increased mobility and nonspecific binding of FoxA1 in mitotic cells. (A) Relative fluorescence intensity (RFI) analysis showing that while GFP-H1o moves threefold more slowly in mitotic chromatin compared with interphase nuclei, GFP-FoxA1 moves 2.5-fold more quickly. Error bars denote standard error of the mean (SEM). Primary FRAP data for RFI analysis. White circles indicate the bleached area. (B) Unique FoxA1 ChIP-seq signals from two to 10 reads per million per 25-bp interval mapped to the left arm of human ch. 7 depicting higher nonspecific, background binding in mitotic cells (red arrows) and many more peaks in asynchronous cells. Input DNA is shown at two to 20 reads per 25-bp interval.

Figure 5.

FoxA1 mutants that perturb specific or nonspecific DNA binding reveal significant nonspecific binding to mitotic chromatin. (A) Crystal structure view of the FoxA DBD depicting residues mutated to perturb specific (NH) and nonspecific (RR) DNA binding (Sekiya et al. 2009). (B) The FoxA1 mutant with impaired nonspecific DNA binding is partially released from mitotic chromosomes, while the FoxA1 mutant with impaired specific DNA binding is mostly retained. (C) ChIP-qPCR assays on transfected HUH7 cells in mitosis showing that the FoxA1-RR nonspecific binding mutant can still recognize the Afp −4.1-kb and Ttr promoter target sites that contain FoxA-binding motifs (shown in the top row, red arrowheads), whereas the FoxA1-NH specific binding mutant cannot. Thus, even when loss of nonspecific binding results in most of the FoxA1 being lost from mitotic chromosomes (B), FoxA1 can bind specifically to target sites in mitotic cell chromatin (C).

Figure 6.

FoxA1 is necessary for timely reactivation of target genes as cells exit mitosis. (A) RT-qPCR analysis of primary transcripts in HUH7 cells treated with siRNAs for FoxA1 (si6689) or a control siRNA blocked in mitosis and released for various time points. Data are normalized to Gapdh and to the “block” time point prior to mitotic release. The first two genes in each row exhibited a net increase in synthesis over time, while the others exhibited an initial burst of synthesis. The top row depicts genes that are bound by FoxA1 in mitosis; these are dependent on FoxA1 for late telophase reactivation. The middle row depicts genes that are bound by FoxA1 only in asynchronous cells, many of which depend on FoxA1 for its initial activation. The bottom row depicts genes that are not bound by FoxA1 and are independent of FoxA1 for early reactivation. See Supplemental Figure 5 for more genes of each type. Error bars denote SEM; asterisks indicate significance by a one-tailed Student's t-test: (*) P < 0.05; (**) P < 0.01. (B) The average nascent transcript induction at 105 min post-mitotic block release is shown as a ratio of that in the presence of the FoxA1 siRNA over that for the control siRNA for genes in the three categories in A and Supplemental Figure 5. Error bars denote SEM, and asterisks indicate significance by a one-tailed Student's t-test: (*) P < 0.05; (**) P < 0.01. A separate Mann-Whitney test, not shown, revealed that the FoxA siRNA selectively perturbed mitotic FoxA1-bound versus unbound to P < 0.00013 and asynchronous-only bound versus unbound to P < 0.022. The data show that FoxA1 facilitates the early reactivation of genes bound in mitosis as well as genes bound solely in interphase cells and does not indirectly enhance the reactivation of genes to which it does not bind.

Figure 7.

Specific and nonspecific modes of FoxA1 binding to the mitotic genome allow rapid reactivation of FoxA target genes during mitotic exit.