Transcriptional interference and transcription through regulatory elements (transcriptional read-through) are implicated in gene silencing and the establishment of DNA methylation. Transcriptional read-through is needed to seed DNA methylation at imprinted genes in the germ line and can lead to aberrant gene silencing by DNA methylation in human disease. To enable the study of parameters and factors influencing transcriptional interference and transcriptional read-through at human promoters, we established a somatic cell culture system. At two promoters of imprinted genes (UBE3A and SNRPN) and two promoters shown to be silenced by aberrant transcriptional read-through in human disease (MSH2 and HBA2) we tested, if transcriptional read-through is sufficient for gene repression and the acquisition of DNA methylation. Induction of transcriptional read-through from the doxycycline-inducible CMV promoter resulted in consistent repression of all downstream promoters, independent of promoter type and orientation. Repression was dependent on ongoing transcription, since withdrawal of induction resulted in reactivation. DNA methylation was not acquired at any of the promoters. Overexpression of DNMT3A and DNMT3L, factors needed for DNA methylation establishment in oocytes, was still not sufficient for the induction of DNA methylation. This indicates that induction of DNA methylation has more complex requirements than transcriptional read-through and the presence of de novo DNA methyltransferases.