A) Genomic structure and nucleosome distribution on wt CLN2pr. The CLN2pr contains three well-positioned nucleosomes (shaded ovals) and NDR (hatched bar). The position of SCBs (red rectangles), TATA box (yellow rectangle) and TSS (arrow) are also shown. B) Nucleosome occupancy on the HO* (upper panel) and CLB2* (lower panel), as well as the inferred nucleosome positioning. HO* / CLB2* are constructed by inserting the CLN2pr NDR sequence into the HOpr / CLB2 ORF at the sites indicated by the arrows. The nucleosome occupancy on wt HOpr and CLB2 ORF are also shown in the corresponding panels. The position “0” on the plot represents the translation start site of HO and CLB2 gene. C) Nucleosome occupancy on the CLN2-HOr (blue curve) in comparison with that on the wt CLN2pr (gray). CLN2-HOr is a CLN2pr variant with the NDR replaced by a HOpr sequence (blue hatched bar). The position “0” on the plot represents the CLN2pr TSS (same for below).

A, B) In vivo (gray) and in vitro (black) nucleosome occupancy on the CLN2pr. The in vitro nucleosome assembly is carried out either by salt dialysis (A) or with purified NapI and ACF (B). C) Nucleosome occupancy on the CLN2pr predicted by a mathematical model from . D) In vivo nucleosome occupancy on the CLN2pr in a strain with over-expressed histones.

A) Construction of the HOr-Seg1–4 sequences. We divided the CLN2pr NDR sequence into 4 segments, each ~80 bp in length, and replaced them back into the CLN2-HOr (), one at a time. B) Nucleosome occupancy and inferred positioning on CLN2-HOr (repeated from ) and HOr-Seg1–4 Seg2–4 each independently forms local NDRs. C) The measured nucleosome occupancy on the HOr-Seg2–4 sequences with mutated (colored curves) or wt Seg2–4 (gray curves). Mutations in the Reb1 or Mcm1 binding sites in the Seg2 (top panel), the two Rsc3 binding sites on Seg3 (middle panel), and the Mcm1 binding site on Seg4 (bottom panel), eliminates the local NDR. See for detailed sequence / factor binding sites / mutation information. D) Summary for all the potential factor binding sites on CLN2pr NDR. The binding sites listed on the top row, but not the bottom row, contribute to the NDR formation. Cluster 1–3 represent three conserved sequence elements on Seg4 that lower the local nucleosome density, but the bound factors are yet to be identified (see text for details). E) ChIP measurement of the enrichment of Reb1, Mcm1 and Rsc3 on the wt CLN2pr and CLN2pr-all* (CLN2pr with mutations in the binding sites of Reb1, Mcm1, Rsc3 and Cluster 1–3). The enrichment is normalized to the background level (bkg). The binding of all three factors to the wt CLN2pr are significantly above the bkg (P value shown in panels), whereas Reb1 and Mcm1 binding to the CLN2pr-all* are eliminated.

A–C) The measured nucleosome occupancy on the CLN2pr-all* (reb1* + mcm1* + rsc3* + cluster 1–3*) (A), CLN2pr-reb1*, -mcm1*, -rsc3* (B) and CLN2pr-part* (reb1* + mcm1* + cluster 1–3*) (C) vs. that on wt CLN2pr (gray curves in A–C). Simultaneous mutations in the Reb1, Mcm1, Rsc3 binding sites and Clusters 1–3 eliminates NDR (CLN2pr-all*); mutations in a subset of the binding sites either have no apparent effect on CLN2pr NDR (CLN2pr-reb1* and -rsc3*), or mildly shrink the NDR (CLN2pr-mcm1* and -part*).

A) Example traces of GFP expression driven by wt CLN2pr (black), CLN2pr-del (gray) and CLN2pr-all* (red). Each trace represents the GFP intensity as a function of time in a single yeast cell over multiple cell generations. The arrows indicate the time points of cell division. In the case of CLN2pr-all*, two traces of the GFP measurements are shown. B) Histogram of the peak expression per cell cycle for the wt, CLN2pr-del and CLN2pr-all*. The wt CLN2pr is “on”, and the CLN2pr-del is “off” every cell cycle. In contrast, CLN2pr-all* switches between “on” and “off” cycles with a ~20% “on” probability. C) Normalized average expression levels of CLN2pr variants with only a subset of binding site(s) mutated. gr* represents the mutation in Gcr1 and Rme1 binding sites. Based on the functions of the factors in transcriptional activation vs. NDR formation, we can separate them into two categories: activator vs. nucleosome depleting factors (NDFs).

A) The number of NDRs that contain NDF consensus site(s) and / or PolyA/T, in comparison with the non-NDR control. The ~1700 NDRs containing at least one NDF binding sites are further dissected based on the number of NDF binding sites as well as the presence of PolyA/T. B) For each NDF (factor A; including Abf1, Reb1, Rap1, Rsc3 and Mcm1), the genome-wide NDRs are divided into three groups based on their sequences. Group1: factor A only, Group2: factor A + other nucleosome-repelling elements, Group3: no factor A. The average nucleosome occupancy of NDRs in these three groups are plotted before (black) and after (red) the deletion of factor A. NDRs in Group2 tends to be more “resistant” upon factor A deletion. For comparison, similar test is applied to factors with little or no nucleosome-depleting activity, including Tbf1 and Cep3. Data size (# of NDRs in Group1–3 for each factor): Abf1: 157, 187, 5725; Reb1:238, 287, 5544; Rsc3: 346, 511, 5212; Rap1: 15, 85, 5969; Mcm1: 29, 76, 5964; Tbf1: 41, 133, 5895; Cep3: 83, 207, 5779).

A–E) Simple energetic model for the formation of NDR. Ovals: bound factors on the DNA. Black curves: basal energy landscape for the nucleosome assembly on naked DNA. Blue, yellow, green and purple dashed curves: local energy penalties against nucleosome assembly generated by the factors. Red curves: the overall energy landscape. When the red curve goes above 0, nucleosome assembly becomes energetically unfavorable and NDR will form. See text for details. F) Reaction pathway for transcription activation on CLN2pr. See text for details.