Fkh1 and Fkh2 control genome-wide initiation dynamics of replication origins
To test whether Fkh1 and Fkh2 influence replication origin function, we examined genome-wide origin-firing using BrdU immunoprecipitation analyzed by DNA sequencing (BrdU-IP-Seq), in cells arrested in early S-phase with hydroxyurea (HU). In this analysis, BrdU peak size is proportional to origin efficiency in HU: early-efficient origins produce large peaks while late and/or dormant origins yield smaller or no peaks (Knott et al., 2009b
). Because Fkh1 and Fkh2 play partially complementary, yet opposing roles in regulation of G2/M-phase regulated genes (Murakami et al., 2010
), we analyzed single as well as double deletion mutants of FKH1
. Furthermore, because the double mutant cells exhibit slow, pseudohyphal growth, which complicates their analysis, we also examined these cells with over-expression of C-terminally truncated FKH2
), which largely restores CLB2
cluster gene regulation (Reynolds et al., 2003
). Consistent with this, we found that expression of Fkh2ΔC in fkh1Δ fkh2Δ
cells suppressed their pseudohyphal growth and restored nearly normal growth rate (Fig. S1A
and data not shown).
In wild-type (WT
) cells, 295 peaks of BrdU incorporation were detected genome-wide ( and Data S1
). Combined deletion of FKH1
had an unprecedented effect on origin activity throughout the genome, with the activities of the archetypal early origins ARS305
being strongly reduced (). Genome-wide, of the 352 origins that were detected to fire in WT
and/or fkh1Δ fkh2Δ
cells, 106 (30%) origins were significantly decreased in activity (Fkh-activated) and 82 (23%) were significantly increased (Fkh-repressed) (Data S1
). Deletion of FKH1
significantly (FDR<0.005) altered the activity of specific origins, with 35 being Fkh-activated and 16 Fkh-repressed, whereas deletion of FKH2
had no significant effect on the replication pattern (, S1B, C
, and Data S1
). Fortuitously, expression of fkh2ΔC
, while complementing the pseudohyphal growth defects due to transcriptional deregulation, did not complement the origin deregulation of fkh1Δ fkh2Δ
cells, with virtually all of the same origins being identified as Fkh-activated (95) or Fkh-repressed (80) (, S1B, C
, and Data S1
). This result demonstrates that the C-terminus of Fkh2 is required for origin regulation, and suggests that the effects on origins are independent of transcriptional regulation by Fkh1 and Fkh2. We took advantage of the ability of fkh2ΔC
expression to complement the transcriptional defects, but not the replication defects, and to improve the growth of the double mutant cells to facilitate further analyses of fkh1Δ fkh2Δ
Figure 1 Analysis of early S-phase BrdU incorporation. A. BrdU incorporation plots of chromosomes III and VI are shown; plot colors and symbols correspond to the strain key above. Origins discussed in the text are boxed. B. Two-dimensional clustering of peak counts (more ...)
Two-dimensional clustering of the Fkh-regulated origins based on their peak sizes allows a global comparison of origin activities in the WT, single and double mutant strains. This analysis reveals the extensive deregulation of fkh1Δ fkh2Δ and fkh1Δ fkh2Δ +pfkh2ΔC cells, the strong similarity between replication patterns in the WT and fkh2Δ cells, and the intermediate phenotype of fkh1Δ cells (). These data indicate that Fkh1 and Fkh2 play a major and complementary role in selecting certain origins for early activation, while repressing the activation of others. Fkh1 is sufficient to maintain normal (early) origin regulation in the absence of Fkh2, whereas Fkh2 only partially compensates for the absence of Fkh1.
To appraise the global relationship between origin activities and regulation by Fkh1 and/or Fkh2 (Fkh1/2), we arranged origins according to their WT activity levels (in HU) and plotted the positions of Fkh-activated and -repressed origins (). Fkh-activated origins were strongly enriched among earlier-firing origins while Fkh-repressed origins were strongly enriched among later-firing (or inefficient) origins (p<0.001, hypergeometric test). These results show that Fkh1 and Fkh2 are largely responsible for differential origin firing dynamics throughout the genome.
To examine in more detail the effect of Fkh1 and Fkh2 on temporal origin-firing dynamics, we analyzed replication throughout an unperturbed, synchronous S-phase. Total DNA content analysis showed similar overall replication kinetics in WT
and fkh1Δ fkh2Δ +pfkh2ΔC
cells (hereon fkh1Δ fkh2ΔC
) (Fig. S2A
). We next used BrdU pulse labeling combined with BrdU-IP analyzed by microarray (BrdU-IP-chip) to analyze origin-firing dynamics. At Fkh-activated ARS305
cells, substantial BrdU incorporation occurred during the 12–24min through 30–42min pulses, and ceased by the 36–48min pulse, consistent with the early and synchronous replication of this origin (). In fkh1Δ fkh2ΔC
cells, however, BrdU incorporation at ARS305
was delayed and reduced in comparison, occurring mainly after replication had ceased in the WT
and numerous other early origins showed similar delay of activity in fkh1Δ fkh2ΔC
cells (Data S2
). These data confirm the results of the analysis with HU and demonstrate that Fkh1/2 are required for the early activation of many origins throughout the yeast genome.
Figure 2 Temporal analysis of DNA replication by BrdU pulse-labeling. A. BrdU incorporation plots of chromosome III and a region of XV are shown. Origins discussed in the text are boxed. B. The matrix shows differences (WT-fkh1Δ fkh2ΔC) in BrdU (more ...)
The data also indicate that Fkh1/2 normally repress the earlier firing of many origins (Data S2
). For example, examination of the late-replicating region of chromosome XV demonstrates that several later-firing origins, such as ARS1520
, initiated replication earlier in the mutant cells (). To address the formal possibility that the observed differences in origin activation timing derive from a change in origin activation efficiency, we performed two-dimensional gel electrophoresis analysis of replication initiation structures of Fkh-activated origin ARS305
and Fkh-repressed origin ARS1520
. Both origins exhibit high efficiency in both WT
and fkh1Δ fkh2ΔC
cells (Fig. S2B
). These data confirm that that Fkh1/2 establish the temporal program of origin activation.
For a global view of the impact of Fkh1/2 regulation on the temporal program, we clustered the Fkh-regulated origins according to their peak-count differences in the HU analysis, and plotted the differences in their levels of BrdU-incorporation between WT and mutant for each interval in the time-course (). This analysis shows global correspondence between the change in origin activity in HU and the change in origin activity in the time course in the fkh1Δ fkh2ΔC cells, with Fkh-activated origins firing earlier and Fkh-repressed origins firing later in WT cells. Thus, Fkh1/2 play a major role in determining the characteristic firing times of replication origins throughout much of the yeast genome.
Fkh-regulation involves establishment of replication timing domains
Comparison of the WT
and mutant chromosomal replication profiles reveals additional features of interest, including even earlier replication of centromere (CEN)-proximal sequences, such that these became the earliest replicating region of each chromosome ( and Data S2
). Plotting CEN-proximal origins (ie, within 25kb) in the time-course clustergram shows that many of these origins initiated earlier in the mutant cells and were among the most strongly affected of the Fkh-repressed origins (). Another striking feature of the mutant replication profiles is the delayed replication of most telomere (TEL)-proximal sequences (Data S2
), particularly those with active origins, as evident on the right arm of chromosome III (). These results further demonstrate the global role of Fkh1/2 in determining genome replication timing and suggest a function in chromosomal organization.
We wondered whether the distribution of Fkh-regulated origins along chromosomes might provide additional clues about their functional organization. Chromosomal plots of Fkh-regulated origins (ignoring non-regulated origins) show frequent, linearly contiguous groups of Fkh-activated and -repressed origins, suggesting a non-random distribution (Fig. S3A
). To test this notion rigorously, we applied a permutation test that determines the likelihood that the contiguous groups are random. The result shows that the distribution of Fkh-activated and -repressed origins is non-random and that origins of each class frequently cluster linearly along the chromosome with other members of their class (p<0.01, Fig. S3B
). Together with the CEN- and TEL-specific effects, these results are consistent with Fkh1/2 establishing domains of replication timing.
Fkh1/2 bind and function in cis to Fkh-activated origins
Fkh1 and Fkh2 exhibit similar DNA sequence binding specificities in vitro
and bind extensively throughout the genome, with significant overlap of binding sites (data not shown and (Harbison et al., 2004
; Hollenhorst et al., 2001
; MacIsaac et al., 2006
). To examine the relationship of Fkh1 and Fkh2 binding with origin regulation, we analyzed the distribution of putative Fkh1 and Fkh2 binding sites within 500bp of Fkh-activated, -repressed, and -unregulated origins (see Methods). This analysis shows that Fkh1 and Fkh2 binding sites are enriched near Fkh-activated origins and depleted near Fkh-repressed origins (, hypergeometric test, p<0.01), as expected if Fkh1/2 act through direct binding near Fkh-activated origins. Fkh1 was most enriched, being ~four-fold enriched at Fkh-activated versus -repressed origins, consistent with a predominant role for Fkh1 rather than Fkh2 in origin regulation as indicated by the single mutant analysis above.
Figure 3 Analysis of Fkh1 and Fkh2 binding sites near origins. A and B. Frequencies of expected and actual Fkh1 (A) and Fkh2 (B) consensus binding sites near Fkh-activated, Fkh-unregulated, and Fkh-repressed origins are shown. C. Frequency distribution plots of (more ...)
The enrichment of Fkh1/2 binding sites near origins may explain the selection of these origins for early activation, however, Fkh1/2 bind near some origins that are not Fkh-activated suggesting that Fkh1/2 binding in the vicinity is not sufficient for origin activation. To determine more precisely how Fkh1 and Fkh2 localize in relation to Fkh-regulated origins, we calculated the distance from each origin’s ARS-consensus sequence (ACS), which binds ORC, to the likeliest Fkh1 and Fkh2 binding site within 500bp and plotted the results as a frequency distribution (see Methods). The distribution reveals extraordinary proximity of Fkh1 and Fkh2 consensus sites to ACSs of Fkh-activated origins, with frequent overlap of the Fkh1/2 binding sites and ACSs (). In contrast, Fkh1 and especially Fkh2 showed poorer alignment and binding density with those few Fkh-repressed origins proximal to Fkh1/2 binding sites. These results suggest that the positioning and/or number of these sites may be important for origin regulation
To test directly whether Fkh1/2 regulate origin function through binding in cis to the affected origin, we mutated two putative Fkh1/2 binding sites near ARS305 (ars305Δ2BS). Combined mutation of these sites significantly reduced BrdU incorporation at ARS305, but not at more distal origins, indicating that Fkh1/2 regulate ARS305 directly through binding in cis (). Crucially, mutation of these binding sites eliminated Fkh1 binding to the ARS305 region without eliminating ORC binding (). These results also eliminate concerns that origin deregulation results from mis-expression of a replication factor(s) in fkh1Δ fkh2ΔC cells. Overall, these results demonstrate that Fkh1/2 binding positively influences origin activity.
Fkh-dependent origin regulation is not correlated with transcription levels or changes
The notion of a mechanistic link between replication origin timing and transcriptional state, together with the well-characterized roles of Fkh1 and Fkh2 as transcriptional regulators, suggested that altered transcription, particularly of genes proximal to Fkh-regulated origins, might explain the altered origin firing. Although expression of Fkh2ΔC suppressed pseudohyphal growth, indicating that normal transcriptional regulation had been at least partially restored, we nonetheless wished to determine whether differences in transcription of genes proximal to the affected origins could account for the differences in origin activity. Accordingly, we analyzed global RNA transcript levels using strand-specific RNA quantification by sequencing (RNA-Seq) and RNA Polymerase II (Pol II) occupancy using chromatin immunoprecipitation analyzed by sequencing (ChIP-Seq) of the Pol II core subunit Rpb3 in WT
and fkh1Δ fkh2ΔC
cells, in unsynchronized cells and cells synchronized in G1-phase, when replication timing is established (Dimitrova and Gilbert, 1999
; Raghuraman et al., 1997
). Up-regulation of CLB2
in G1-phase fkh1Δ fkh2ΔC
cells, which is consistent with the role of Fkh1 in CLB2
repression, and significant overlap between genes identified by the different methods validated both analyses (Table S1
). A permutation test indicates that genes deregulated in fkh1Δ fkh2ΔC
cells are not significantly co-localized with or proximal to Fkh-regulated origins (see Methods). We also plotted RNA transcript levels and Rpb3 occupancy, as well as their differences in fkh1Δ fkh2ΔC
cells, within 10kb of Fkh-regulated origins (). Visual inspection of these plots show no obvious correlation with the effects on origin activities, regardless of the magnitude or directionality (positive or negative) of effect, the orientation of the immediately flanking genes, or the cell cycle stage. Linear regression analysis also shows no consistent correlation between the effects on origin activity and the expression levels of the immediately flanking genes (see Methods). These findings demonstrate that origin regulation by Fkh1/2 does not involve proximal changes in transcription.
Figure 4 Transcription analysis surrounding Fkh-regulated origins in unsynchronized and G1-synchronized cells. RNA-Seq (A) and Rpb3 ChIP-Seq (B) read counts of WT, fkh1Δ fkh2ΔC, and WT-fkh1Δ fkh2ΔC differences (Δ), within (more ...)
Cdc45 preferentially associates with Fkh-activated origins in G1-phase
We wondered whether Fkh1/2 regulate replication timing by modulating the binding of replication factors to origins. To determine whether Fkh1/2 influence ORC binding or MCM loading, we used ChIP analyzed by microarray (ChIP-chip) to examine ORC binding in unsynchronized cells and Mcm2+4 binding in G1-synchronized cells. This results show no significant differences in ORC or Mcm2+4 binding between WT and fkh1Δ fkh2ΔC cells (), contrary to the idea that Fkh1/2 affect origin-firing by modulating ORC binding or pre-RC assembly.
Figure 5 Genome-wide binding of replication initiation factors to Fkh-regulated origins. A. M-values from ChIP-chip analysis of ORC, Mcm2+4, and Cdc45 at Fkh-regulated origins (in rows) are arranged by differences (WT-fkh1Δ fkh2ΔC) in BrdU incorporation (more ...)
Origin initiation requires the DDK-dependent recruitment of Cdc45 to pre-RCs. However, Cdc45 associates specifically, albeit relatively weakly, with several early replication origins in G1-phase (prior to DDK activation), presaging their characteristic early S-phase activity (Aparicio et al., 1999
). This suggests that these origins gain an early advantage (by G1-phase) in their ability to recruit Cdc45 to enable early initiation. Examination of Cdc45 binding by ChIP-chip shows Cdc45 association with many early origins, including Fkh-activated origins, such as ARS305
, and a number of CEN-proximal origins (). Of 29 origins that bind Cdc45 in WT
G1-phase cells, 15 are Fkh-activated and 14 are CEN-proximal (on 11 CENs), while only one is Fkh-repressed. Strikingly, in the fkh1Δ fkh2ΔC
cells, Cdc45 binding is lost from the Fkh-activated origins, which become significantly later firing, leaving only 14 origins binding Cdc45 (). Of these 14, 12 are CEN-proximal, which as shown above, remain early firing. Thus, Cdc45 origin-binding in G1-phase is robustly associated with early initiation. These findings support the idea that Fkh1/2 influence origin function by regulating access to the pool of replication factors such as Cdc45, whereas CEN-proximal origins have access to Cdc45 independently of Fkh1/2.
Fkh1/2 are required for selective clustering of Fkh-activated origins in G1-phase
The organization of selected origins into subnuclear domains or replication foci by Fkh1/2 may explain their preferential access to limiting or sequestered initiation factors like Cdc45. In accord with this, a global analysis of intra- and inter-chromosomal interactions of the yeast genome using a variation of 4C (C
hip) suggests that early origins cluster in G1-phase (Duan et al., 2010
). We analyzed this origin interaction data to determine whether origin clustering was associated with Fkh-regulation and/or Cdc45 binding in G1-phase. Two-dimensional clustering based on origin interaction frequencies resulted in two main clusters of interacting origins, with 89 and 92 origins, respectively (). One cluster contains most of the Cdc45-bound origins, the most statistically significant Fkh-activated origins, and CEN-proximal origins. This cluster also contains earlier-firing origins on average than the other main cluster and is depleted of non-CEN proximal, Fkh-repressed origins (hypergeometric test, p<0.005). These findings suggest that Fkh-regulation involves selective origin clustering.
Figure 6 Chromosome-conformation capture analyses of origin interactions. A. Two-dimensional clustering of origin-origin interaction frequencies is shown, with origins in columns and rows of the matrix. Columns to the right indicate Cdc45 ChIP-chip binding, average (more ...)
To test whether Fkh1/2 have a role in origin clustering, we used 4C to analyze the trans
associations of Fkh-activated origin ARS305
with other genomic sequences (for scheme, see Fig. S5A
). We validated this analysis by comparing overlap between experimental replicates of WT
and mutant cells, with and without crosslinking, and by analyzing the number of intra- versus inter-chromosomal interactions detected (Fig. S5B
). As expected, and consistent with the results of (Duan et al., 2010
), intrachromosomal interactions were enriched versus interchromosomal interactions (p<0.001). We detected 48 ARS305
-interacting loci in both WT
replicates (of 71 and 72 in the replicates), and 41 ARS305
-interacting loci in both fkh1Δ fkh2ΔC
replicates (of 164 and 189 in the replicates) (). The larger number of detected interactions with lower overlap between them in the fkh1Δ fkh2ΔC
replicates is consistent with a decrease in specificity of ARS305
interactions in the mutant cells. Most of the 48 sites in WT
cells were not detected in the mutant cells, indicating that their interaction with ARS305
is Fkh1/2-dependent. For example, ARS305
interacted with ARS607
(as shown previously (Duan et al., 2010
)) in both WT
replicates and in neither fkh1Δ fkh2ΔC
replicate (), indicating that Fkh1/2 are required for interaction in G1-phase between these early-firing, Fkh-activated origins. These results indicate that Fkh1/2 play a role in determining the long-range chromatin contacts made by ARS305
, and support the idea that Fkh1/2 function in origin regulation through origin clustering.