The model formulated by Tatjana Degenhardt, Katja N. Rybakova, Aleksandra Tomaszewska, Martijn J. Mone , Hans V. Westerhoff,

Frank J. Bruggeman, and Carsten Carlberg on transcription cycling is

generic in essence and does not depend on the particular set of data in the original article. The model provides an explanation for many independent experimental observations (list of articles provided below):

 

1.    Cyclic changes in the occupancy by proteins and the state of nucleosome modifications/positioning of eukaryotic promoters at the population level in various cell models (all of the articles listed below). The periods of the oscillations range from 40 min (Metivier et al., 2008; Metivier et al., 2003) to 90 min (Kang et al., 2002). All of these can be reproduced by the model, with realistic protein binding constants and protein concentrations.

 

2.    Desynchronization of the cycling over time, manifesting in decrease of peaks over time, can be seen in data by (Karpova et al., 2008) (Fig. 2). The theoretical exploration of the subject was presented in the appendix of the paper (Fig. S11)

 

3.    Cycling on promoters that are active prior to activation by TF (such as observed on the OPN promoter (Kim et al., 2005) and (Saramaki et al., 2009). The cells are still sufficiently synchronized, as at low activity most of the promoters are in the inactive state, as demonstrated directly in the paper (Fig. S7CD)

 

4.    Cycling at the mRNA level, provided that mRNA degradation is a multi-step process with time scale comparable to cycling (Karpova et al., 2008) (Fig. 3)

 

5.    Production of multiple mRNAs per cycle, strongly suggested by data of Karpova et al. (Fig. 4). This is also in accordance with the observation of large and infrequent mRNA bursts in eukaryotic cells (Raj et al., 2006)

 

6.    Blocking part of the cycle causes the system to get stuck in that part of the cycle (data from Kang et al, 2002) (Fig. 5).

 

7.    Cycling being possible if a distant TF binding site is involved and also observed with looping of the promoter (Fig. 5)


8.   

Fig.1: Population level cycling with different time periods. TOP: 40 min period (Metivier et al., 2008; Metivier et al., 2003), free protein concentration in the model is 750 molecules/nucleus, MIDDLE: period of 75 min (Kim et al., 2005), free protein concentration in the model is 400 molecules/nucleus, BOTTOM: period of 90 min (Kang et al., 2002), free protein concentration in the model is 333 molecules/nucleus.

 

Fig 2. Desynchronization between cells leads to a decrease in peak height. TOP: experimental data on TF cycling on a yeast promoter (Karpova et al., 2008), BOTTOM: simulation of model with similar cycling period.

 

 

 

Fig 3. Cycling on the level mRNA for the yeast CUP1 gene TOP: experimental data (Karpova et al., 2008), BOTTOM: simulation of model with similar cycling period of 50min, mRNA degradation is multistep process at an average time of 8 min.

 

 

Fig 4. Production of multiple mRNAs per cycle. TOP: experimental data from Karpova et al, measured in arbitrary units; no apparent RNA degradation during experiment due to introduction of MS2-repeats at the 3 end, MIDDLE: model reproducing the data (with negligible degradation), BOTTOM: same model with degradation as in simulation on population level displays burst of mRNA production over time

 

 

 

Fig 5. Effects of blocking of one of the irreversible steps in the cycle (Kang et al., 2002), TOP: Model reproducing pattern of binding observed in experiment (Black – androgen receptor (AR), Red – polymerase). BOTTOM: Model reproducing the experiment with inhibition of proteosome complex that shows lack in decrease of AR binding. (solid line – control, dotted line – with inhibitor).

 

 

Fig. 6. Cycling of VDR (blue), pPolII (red) and loop between TSS and RE for data by (Saramaki et al., 2009).


 

Supporting data:

 

1. Metivier et al (2003, 2008)

 

Gene: Ps2, inactive prior to ligand addition (additionally synchronized by alpha-amanitin)

TF and promoter architecture: activated by ER, RE and TSS very close (not distinguished in ChIP)

System: human breast epithelium line

Main observations: Cycling of ER, pPolII and another 38 proteins (including histone modifiers, re-modellers , proteosome components, DNA repair and DNA modifiers), 4 histone modifications and several DNA methylations shown to cycle with 2 periodicities 40 (most) and 80 (H4R3me, some of DNA methylations, TBP TAFIIA).

Additionally demonstrated:

cycles in accessibility of TSS and ERE by nucleosome positioning .

mRNA – run-on (initiation rate) shown to fluctuate correspondingly

 

2. Karpova et al, 2008

Gene: CUP1, inactive prior to Cu2+ addition

TF and promoter architecture: activation by Ace2p, multiple binding sites close to TSS (not distinguished in ChIP or microscopy)

System: yeast cells

Main observations: Cycling of Ace2p with microscopy (GFP-tagged) and ChIP: approx. 45-50 min periodicity, peaks 10, 60 and 100min, mRNA cycling with same periodicity (and strangely same peak times – check primer locations)

 

Additionally demonstrated: measurements on single cell correspond to population level (ChIP),

TF binding peaks become lower (desynchronization)

Data suggesting that more than 1 mRNA per cycle is produced (but not conclusive)

 

3. Kang et al, 2005

 

Gene: PSA (seems to be active before addition of testosterone as mRNA is present)

TF and promoter architecture activated by AR, ARE very close to TSS (no distinction in ChIP)

System: human prostate cell line

Main observations: Cycling of AR, PolII, GRIP1, CBP, AcH3, proteosome subunit 19S shown – 90min frequency first peak 45min for AR, 60 min for PolII and rest

Additionally demonstrated: proteosome block stops promoter deactivation – constant AR and 19S binding

 

4. Kim et al, 2002

 

Gene: promoter Cyp24 (inactive before vitD addition) and OPN (active before vitD addition)

TF and promoter architecture activated by VDR, for Cyp24VDRE close to TSS (not distinguished in ChIP), for OPN VDRE far from TSS (only RE assessed)

System: mouse osteoblast cell line

Main observations: Shown cyclic VDR, PolII and DRIP205 and SRC-2 binding with frequency of 75min (first peak at 45min) for all

 

5. Saramaki et al 2009

 

Gene: p21 (active before addition of vitD)

TF and promoter architecture activated by VDR, 3 VDREs far from to TSS (distinguished in ChIP)

System: human breast cancer cell line

Main observations: Cycling of pPoll, VDR and MED1 shown convincingly frequency 60min (first peak around 30min for both) on TSS and one of the VDR

 

Additionally demonstrated: Looping of RE1,2 to TSS with same frequency and peaks

Possibly mRNA cycling peaks at 60 and 105min

 

List of references

 

Kang, Z., Pirskanen, A., Janne, O.A. and Palvimo, J.J. (2002) Involvement of proteasome in the dynamic assembly of the androgen receptor transcription complex. J Biol Chem, 277, 48366-48371.

Karpova, T.S., Kim, M.J., Spriet, C., Nalley, K., Stasevich, T.J., Kherrouche, Z., Heliot, L. and McNally, J.G. (2008) Concurrent Fast and Slow Cycling of a Transcriptional Activator at an Endogenous Promoter. Science, 319, 466-469.

Kim, S., Shevde, N.K. and Pike, J.W. (2005) 1,25-Dihydroxyvitamin D3 stimulates cyclic vitamin D receptor/retinoid X receptor DNA-binding, co-activator recruitment, and histone acetylation in intact osteoblasts. J Bone Miner Res, 20, 305-317.

Metivier, R., Gallais, R., Tiffoche, C., Le Peron, C., Jurkowska, R.Z., Carmouche, R.P., Ibberson, D., Barath, P., Demay, F., Reid, G., Benes, V., Jeltsch, A., Gannon, F. and Salbert, G. (2008) Cyclical DNA methylation of a transcriptionally active promoter. Nature, 452, 45-50.

Metivier, R., Penot, G., Hubner, M.R., Reid, G., Brand, H., Kos, M. and Gannon, F. (2003) Estrogen receptor a directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell, 115, 751-763.

Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. and Tyagi, S. (2006) Stochastic mRNA Synthesis in Mammalian Cells. PLoS Biol, 4, e309.

Saramaki, A., Diermeier, S., Kellner, R., Laitinen, H., Vas¤nen, S. and Carlberg, C. (2009) Cyclical Chromatin Looping and Transcription Factor Association on the Regulatory Regions of the p21 (CDKN1A) Gene in Response to 1,25-Dihydroxyvitamin D3. Journal of Biological Chemistry, 284, 8073-8082.