1. Supplementary Figure 1. mRNA induction/repression kinetics of HXK1, GAL1::FMP27 and INO1 (A) RT-qPCR analysis of HXK1 kinetic response to Galactose induction and Glucose repression in an mlp1Δ strain. (B) RT-qPCR analysis of GAL1::FMP27 kinetic response to Galactose induction and Glucose repression in a wild type strain. (C) RT-qPCR analysis of GAL1::FMP27 kinetic response to Galactose induction and Glucose repression in an mlp1Δ strain. (D) RT-qPCR analysis of INO1 kinetic response to –Inositol induction and +Inositol repression. (E) Pol II ChIP profile on HXK1 upon initial Galactose induction and subsequent Glucose repression. The Glucose repression time-points are those taken prior to galactose re-induction in the time-course experiments. D A C B E Tan-Wong_US 18232_Suppl.Fig.1

2. Supplementary Figure 2A. HXK1 3C controls for WT and mlp1Δ strains. (A)The following controls were performed for HXK1 MseI 3C on the WT and mlp1Δ strains – (I) Loading control PCR on HXK1 using the amplicon ‘D’ in Fig1A; (II) Positive control for MseI 3C processing on SEN1; (III) 3C negative controls probing promoter-internal fragment interactions (primer combination 1-2) and (IV) promoter-distal fragment interactions (primer combination 1-4) to control for specificity of the gene loop (V) (primers 1-3 as in Fig. 1A). All 3C PCRs were performed alongside a PCR positive control using MseI digested and ligated genomic DNA to control for all possible ligation combinations and primer efficiency. 1’5’10’20’40’60’120’ GAL induction (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) A(I) HXK1 loading control WTmlp1Δ 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’120’ GAL induction A(III) HXK1 primers 1-2 (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) 1’5’10’20’40’60’120’ GAL induction A(II) SEN1 3C control (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) A(IV) HXK1 primers 1-4 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’120’ GAL induction 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’120’ GAL induction A(V) HXK1 primers 1-3 1’5’10’20’40’60’4.5hr GLU repression +ve 1’5’10’20’40’60’120’ GAL induction +ve 1’5’10’20’40’60’4.5hr GLU repression +ve 1’5’10’20’40’60’120’ GAL induction (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) 1’5’10’20’40’60’120’ GAL induction GLU-GAL GLU-GAL-GLU 60’-GAL GLU-GAL-GLU 4.5hr-GAL 1’5’10’20’40’60’120’ GAL induction 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’4.5hr GLU repression +ve (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) 1’5’10’20’40’60’120’ GAL induction +ve 1’5’10’20’40’60’4.5hr GLU repression +ve 1’5’10’20’40’60’120’ GAL induction +ve 1’5’10’20’40’60’120’ GAL induction (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’4.5hr GLU repression 1342 Tan-Wong_US 18232_Suppl.Fig.2A

3. B(I) GAL1::FMP27 loading control 1’5’10’20’40’60’120’ GAL induction (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’120’ GAL induction 1’5’10’20’40’60’4.5hr GLU repression (GLU-GAL) (GLU-GAL-GLU 60’-GAL) (GLU-GAL-GLU 4.5hr-GAL) B(II) FMP27 primers 1-2 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’4.5hr GLU repression 1’5’10’20’40’60’120’ GAL induction 1’5’10’20’40’60’120’ GAL induction Supplementary Figure 2B. GAL1::FMP27 3C controls. (B)Controls for GAL1::FMP27 EcoRI 3C – (I) Loading control PCR on GAL1::FMP27; (II) Positive control for EcoRI 3C processing using FMP27 primers 1-2. Negative controls were performed along FMP27, probing promoter-internal fragment interactions (primer combination 1-3) to control for specificity of the gene loop (primers 1-4 as in Fig. 3A). Data is not shown since, as expected, no 3C signals were obtained and these negative controls have been published previously (1). All 3C PCRs were performed alongside a PCR positive control using EcoRI digested and ligated genomic DNA to control for all possible ligation combinations and primer efficiency. WTmlp1Δ 1 2 3 4 Tan-Wong_US 18232_Suppl.Fig.2B

4. Supplementary Figure 2C. HXK1 3C controls for isogenic WT and sua7-1 strains. (A)The following controls were performed for HXK1 MseI 3C on the isogenic wildtype and sua7-1 strains – (I) Loading control PCR on HXK1 using the amplicon ‘D’ in Fig1A; (II) Positive control for MseI 3C processing on SEN1; (III) 3C negative control probing promoter-internal fragment interaction (primer combination 1-2) to control for specificity of the gene loop (primers 1-3 as in Fig. 5A). All 3C PCRs were performed alongside a PCR positive control (‘+’) using MseI digested and ligated genomic DNA to control for all possible ligation combinations and primer efficiency. A(I) A(III) HXK1 primers 1-2 A(II) 1342 GLU o/n GAL 60’ GLU 60’ WT GLU o/n GAL 60’ GLU 60’ sua7-1 + GLU o/n GAL 60’ GLU 60’ WT GLU o/n GAL 60’ GLU 60’ sua7-1 + GLU o/n GAL 60’ GLU 60’ WT GLU o/n GAL 60’ GLU 60’ sua7-1 + Tan-Wong_US 18232_Suppl.Fig.2C

5. Supplementary Figure 2D. INO1 3C controls. (C)Controls for INO1 HaeIII 3C – (I) Loading control PCR on INO1; (II) Positive control for EcoRI 3C processing using INO1 primers I1-I2; (III) 3C negative control probing promoter-internal fragment interactions (primer combination I2-I4) to control for specificity of the gene loop (IV; primers I1-I6) as in Fig. 4A). All 3C PCRs were performed alongside a PCR positive control (‘+’) using HaeIII digested and ligated genomic DNA to control for all possible ligation combinations and primer efficiency. (II) I2 – I1 o/n OFF 1hr ON 1hr OFF 1hr ON 12hr OFF 1hr ON + (I) Loading control o/n OFF 1hr ON 1hr OFF 1hr ON 12hr OFF 1hr ON + (III) I2 – I4 o/n OFF 1hr ON 1hr OFF 1hr ON 12hr OFF 1hr ON + (IV) I2 – I6 o/n OFF 1hr ON 1hr OFF 1hr ON 12hr OFF 1hr ON + I1 I4 Tan-Wong_US 18232_Suppl.Fig.2D

6. Supplementary Figure 3. sua7-1 re-induction analysis Induction/re-induction time-course data for sua7-1 and its isogenic wildtype, showing a comparison between induction following long-term (overnight) repression (white bars) and re- induction following short-term (60min.) intervening repression. Consistent with the data in Figure 5C, wildtype shows faster re-induction memory, whereas this memory is absent in the sua7-1 mutant. Tan-Wong_US 18232_Suppl.Fig.3 0 20 40 60 80 100 120 140 160 180 WTsua7-1 mRNA levels GLU o/n - GAL 1hr GLU-GAL-GLU60'-GAL1hr

7. Supplementary Figure 4. Analysis of GAL:FMP27 long-term induction kinetics GAL1:FMP27 RT-PCR analysis showing long-term mRNA induction profile in a WT strain. The top and bottom graphs are similar, except that the bottom graph contains a wider y-axis scale as it includes the additional 5hr and 8hr time-points. It is observed that stable mRNA levels increase greatly between 3hrs and 5hrs, with the ‘memory’ conditions still providing the highest levels. A drop in mRNA levels is observed between 5hrs and 8hrs, indicating possible transcriptional auto-regulation or mRNA stability regulation at these much later time-points. GLU-GAL GLU-GAL-GLU60’-GAL GLU-GAL-GLU4.5hr-GAL Tan-Wong_US 18232_Suppl.Fig.4

8. Supplementary Figure 5. Nup2 deletion has no effect on transcriptional memory. GAL1::FMP27 RT-qPCR data showing kinetics of Galactose induction and re-induction in wildtype and nup2Δ. Time-points were taken at 1hr (empty box) and 2hrs (grey box) of first- round galactose induction (after overnight growth in glucose) and at 1hr (hashed box) and 2hrs (black box) of second-round galactose re-induction (after an intervening 1hr of glucose repression). The nup2Δ strain shows transcriptional memory effects similar to wildtype, consistent with the 3C data in Fig. 1D showing maintenance of the gene memory-loop in nup2Δ. O/N Glucose Galactose 1hr Glucose 1hr Galactose 2hr Galactose re- induction 1hr Galactose re- induction 2hr Tan-Wong_US 18232_Suppl.Fig.5 0 20 40 60 80 100 WT nup2  mRNA levels GAL 1hr GAL 2hr GAL - GLU 1hr - GAL 1hr GAL - GLU 1hr - GAL 2hr

9. Supplementary Figure 6. ADH1 transcript levels remain stable during glucose/galactose treatment and between WT and sua7-1 strains Control experiment with ADH1 mRNA normalized to PYK1 transcripts. ADH1 mRNA levels remain largely stable between WT and sua7-1 strains and during glucose/galactose treatments, confirming its reliable use as a normalization control in our experimental conditions for the HXK1 and GAL1:FMP27 RT-qPCR analysis. Tan-Wong_US 18232_Suppl.Fig.6 ADH1 control 0 20 40 60 80 100 WT GLU o/n WT GAL 10' WT GAL 40' WT GAL 1hr WT GAL 2hrs sua7-1 GAL 1hr ADH1 mRNA levels (normalized to PYK1)

10. Supplementary Figure 7. TBP is maintained at GAL1:FMP27 during 1hr glucose repression in wildtype, but not in mlp1Δ Anti-TBP ChIP performed on wildtype (WT) and mlp1Δ strains at the GAL1::FMP27 promoter at the time-points of galactose growth (GAL) and a subsequent 60min glucose repression (GLU60’). This latter time-point is that at which memory gene loops and transcriptional memory are maintained in wildtype but not in mlp1Δ. As seen here, TBP is still abundantly present during 1hr glucose repression in wildtype, but decreases in an mlp1Δ mutant strain in which memory is lost. Tan-Wong_US 18232_Suppl.Fig.7 TBP ChIP 0.0 0.5 1.0 1.5 2.0 GALGLU WT mlp1 

11. Supplementary Figure 8A. A difference in Gal1 protein levels is not the cause of the memory loss in mlp1Δ mutants. Western blot analysis showing Gal1-GFP and Actin (as loading control). Gal1 protein levels are not detectible in the wildtype and mlp1Δ strains, even during the intervening repression time- point of ‘GLU 60’, during which wildtype cells retain memory, but mlp1Δ cells do not. We presume that the 60’ Galactose induction period prior to short term 60’ Glucose repression or longer term 4.5 hrs repression is insufficient time to accumulate Gal1-GFP protein. These results argue for a Mlp1 (and associated gene-loop) specific memory effect, and against a solely cytoplasmic effect of Gal1 in conferring memory (Zacharioudakis et al, 2007). O/N GlucoseGalactose 60’ Glucose 60’ Glucose 4.5hrs WTmlp1Δ GAL o/n WTmlp1ΔWTmlp1ΔWTmlp1Δ GLU 60’GLU 4.5hrsGLU o/n WTmlp1Δ Untagged GAL o/n Actin (~42kDa) Gal1-GFP (~85kDa) Tan-Wong_US 18232_Suppl.Fig.8A

12. 50 100 untagGlu o/n60’ Glu4.5 hr GluGal o/n % population of flourescence wild type mlp1 ∆ Supplementary Figure 8B. FACS analysis at representative time points showing single cell analysis of Gal1-GFP fluorescence measurements during repression time points. Gal1-GFP-tagged yeast strains expressing GFP fluorescence by FACs analysis shows similar fluorescence levels between wild type and mlp1Δ strains. i) Diagram of fluorescence measurement (Y-axis: FL1-H) against 30000 counts of yeast cells (X-axis: SSC-H). Left panel is the untagged strain (untag) while right panel shows the fluorescence of Gal1-GFP-tagged yeast grown in galactose overnight (Gal o/n). ii) Histogram of FACS analysis showing mean Gal1-GFP fluorescence values, over 3 biological repeats, at representative time points taken from wild type and mlp1∆ grown continuously in glucose o/n (Glu o/n), washed in water before activating in galactose media for 60 minutes. The strains were then subjected to repression in glucose media for 60 minutes (60’ Glu) and and 4.5 hours (4.5hr Glu). Note that as for the western blot analysis (Supplemenary Figure 8A) no fluorescent cells were detected during the short Glucose repression period (memory) arguing against Gal1-GFP effects in this process. O/N GlucoseGalactose 60’ Glucose 60’ Glucose 4.5hrs i) ii) untag Gal o/n Tan-Wong_US 18232_Suppl.Fig.8B

13. Supplementary Figure 9. Western blot control of Mlp1-GFP tagged WT and sua7-1 strains Western blot control of isogenic wildtype and sua7-1 strains (3 clones each shown here) that were Mlp1-GFP tagged using homologous recombination. The western blot has been probed with α-GFP and α –Actin (loading control). A Gal1-GFP tagged strain was used as a control for the GFP antibody; a Mlp1-GFP strain from Feuerbach et al (2003) was used as a positive control; and an untagged strain was used as a negative control. Tan-Wong_US 18232_Suppl.Fig.9

14. Supplementary Table 1. Primers used in ChIP, 3C and RT-qPCR analysis Tan-Wong_US 18232_Suppl.Table.1